Nucleic acids encoding anti-il-23 antibodies

ABSTRACT

The present invention relates to blocking, inhibiting, reducing, antagonizing or neutralizing the activity of IL-17, IL-23 via it&#39;s p19 subunit or both IL-17 and IL-23 (via p19). IL-17 and IL-23 are cytokines that are involved in inflammatory processes and human disease.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.12/870,199, filed Aug. 27, 2010, which is a continuation of U.S. patentapplication Ser. No. 11/762,738, filed Jun. 13, 2007, now U.S. Pat. No.7,790,862, which claims the benefit of U.S. Provisional Application Ser.No. 60/804,602, filed Jun. 13, 2006, U.S. Provisional Application Ser.No. 60/824,665, filed Sep. 6, 2006, U.S. Provisional Application Ser.No. 60/828,277, filed Oct. 5, 2006, and U.S. Provisional ApplicationSer. No. 60/891,410, filed Feb. 23, 2007, all of which are hereinincorporated by reference.

FIELD OF THE INVENTION

The present invention relates generally to the identification andisolation of antagonists to IL-17 and IL-23 (via p19) and methods ofusing the same.

BACKGROUND OF THE INVENTION

The immune system protects individuals from infective agents (e.g.viruses, bacteria, and multi-cellular organisms), as well as from cancerand neoplasms. The immune system includes many lymphoid and myeloid celltypes such as neutrophils, monocytes, macrophages, dendritic cells(DCs), eosinophils, T cells, and B cells. These cells are capable ofproducing signaling proteins known as cytokines. Cytokines are soluble,small proteins that mediate a variety of biological effects, includingthe induction of immune cell proliferation, development,differentiation, and/or migration, as well as the regulation of thegrowth and differentiation of many cell types (see, for example, Arai etal., Annu. Rev. Biochem. 59:783 (1990); Mosmann, Curr. Opin. Immunol.3:311 (1991); Paul and Seder, Cell 76:241 (1994)). Cytokine-inducedimmune functions can also include an inflammatory response,characterized by a systemic or local accumulation of immune cells.Although they do have host-protective effects, these immune responsescan produce pathological consequences when the response involvesexcessive and/or chronic inflammation, as in autoimmune disorders (suchas multiple sclerosis) and cancer/neoplastic diseases (Oppenheim andFeldmann (eds.) Cytokine Reference, Academic Press, San Diego, Calif.(2001); von Andrian and Mackay New Engl. J. Med. 343: 1020 (2000);Davidson and Diamond, New Engl. J. Med. 345:340 (2001); Lu et al, Mol.Cancer Res. 4:221 (2006); Dalgleish and O'Byrne, Cancer Treat Res. 130:1(2006)).

Proteins that constitute the cytokine group include interleukins,interferons, colony stimulating factors, tumor necrosis factors, andother regulatory molecules. For example, human interleukin-17A (alsoknown as “IL-17”) is a cytokine which stimulates, for example, theexpression of interleukin-6 (IL-6), intracellular adhesion molecule 1(ICAM-1), interleukin-8 (IL-8), granulocyte macrophagecolony-stimulating factor (GM-CSF), and prostaglandin E2, and plays arole in the preferential maturation of CD34+ hematopoietic precursorsinto neutrophils (Yao et al., Immunol. 155:5483 (1995); Fossiez et al.,J. Exp. Med. 83:2593 (1996)). As another example, human interleukin-23(also known as “IL-23”) is a cytokine which has been reported to promotethe proliferation of T cells, in particular memory T cells and cancontribute to the differentiation and/or maintenance of Th17 cells.

Accordingly, the demonstrated in vivo activities of cytokines and theirreceptors illustrate the clinical potential of, and need for, othercytokines, cytokine receptors, cytokine agonists, and cytokineantagonists. For example, demonstrated in vivo activities of thepro-inflammatory cytokine family illustrates the enormous clinicalpotential of and need for antagonists of pro-inflammatory molecules suchas IL-17A and IL-23.

DETAILED DESCRIPTION OF THE INVENTION

The present invention addresses these needs by providing antagonists topro-inflammatory cytokines IL-17 (also interchangeably referred to as“IL-17A” herein) (SEQ ID NOS:1 & 2) and the p19 subunit (SEQ ID NOS:3 &4) of IL-23 (SEQ ID NOS:5 & 6).

IL-17 is a cytokine which stimulates the expression of IL-6, ICAM-1,IL-8, GM-CSF, and prostaglandin E2, among others, and plays a role inthe preferential maturation of CD34+ hematopoietic precursors intoneutrophils (Yao et al., J. Immunol. 155:5483 (1995); Fossiez et al., J.Exp. Med. 183:2593 (1996)).

IL-23 is a heterodimeric cytokine composed of a unique subunit, p19(herein referred to interchangeably as “IL-23”, “p19” and “IL23/p19”),and the p40 subunit, which is shared with interleukin-12 (IL-12)(Oppmann, Immunity 13:715 (2000)). IL-23 has been found to stimulate theproduction and/or maintenance of IL-17 A and F from activated CD4− Tcells in what has now been termed as a “new” T-helper (Th) subset,designated Th17. A review of IL-23 cytokine and receptor biology isreviewed in Holscher, Curr. Opin. Invest. Drugs 6:489 (2005) andLangrish et al. Immunol Rev. 202:96 (2004). Similar to Th1 and Th2lineages, Th17 cells have most likely evolved to provide adaptiveimmunity to specific classes of pathogens, such as extracellularbacteria. However, inappropriate Th17 responses have been stronglyimplicated in a growing list of autoimmune disorders, including multiplesclerosis, rheumatoid arthritis, inflammatory bowel disease, andpsoriasis.

In fact, both IL-17 and IL-23 have also been reported to play importantroles in many autoimmune diseases, such as multiple sclerosis,rheumatoid arthritis, Crohn's disease, and psoriasis. Both IL-23 andIL-17 are overexpressed in the central nervous system of humans withmultiple sclerosis and in mice undergoing an animal model of multiplesclerosis, experimental autoimmune encephalomyelitis (EAE). Theoverexpression is observed in mice when the EAE is induced by eithermyelin oligodendrocyte glycoprotein (MOG) 35-55 peptide- or proteolipidpeptide (PLP). Furthermore, neutralization of either IL-23/p19 or IL-17results in amelioration of EAE symptoms in mice (Park et al, Immunol.6:1133 (2005); Chen et al, J Clin Invest. 116:1317 (2006)).

It has also been demonstrated that IL-17 and Th17 cells can be producedfrom IL-23-independent sources, and the in vivo development of an IL-17effector response has been shown to be IL-23-independent (Mangan et al,Nature 441:231 (2006)). Neutralization of IL-23 would theoreticallyeliminate existing IL-17 producing cells, but would not completelyprevent the development of new Th17 cells.

An important regulator of IL-23 independent production of Th17 istransforming growth factor-beta (TGF-b). It has been repeatedlydemonstrated that TGF-b (including TGF-b1) is critical for commitment toTh17 development, independent from IL-23 (Mangan et al, Nature. 441:231(2006)). This is further supported by the fact that development of Th17cells is markedly impaired in mice deficient in TGF-b1 (Mangan et al,Nature. 441:231 (2006)). The idea that TGF-b1 appears to have both anti-and pro-inflammatory roles may be due in part to the fact that it caninduce the expression of the pro-inflammatory cytokine IL-17 and Foxp3(the transcription factor for the CD4+CD25+ regulatory T cellpopulation), but by distinct CD4+ T cell subpopulations (Mangan et al,Nature. 441:231 (2006)).

Receptors that bind cytokines are typically composed of one or moreintegral membrane proteins that bind the cytokine with high affinity andtransduce this binding event to the cell through the cytoplasmicportions of the certain receptor subunits. Cytokine receptors have beengrouped into several classes on the basis of similarities in theirextracellular ligand binding domains. IL-17 mediates its effects throughinteraction with its cognate receptor, the IL-17 receptor (IL-17R) aswell as IL-17RC. The IL-23 and IL-12 receptors share a subunit,IL-12Rb-1 that pairs with unique, inducible components, IL-23R andIL-12Rb-2, respectively, which in turn is responsible for receptorresponsiveness. One of the actions of TGF-b in this respect is toupregulate IL-23R expression, which in turn, induces responsiveness toIL-23.

The present invention concerns the inhibition of both of theseproinflammatory cytokines, IL-17 and IL-23/p19. The present invention isbased on the surprising discovery that antagonizing both IL-23 (via p19)and IL-17 is more effective therapeutically than neutralization of IL-23alone (either via p19 or p40) or IL-17 alone and thus, necessary for theeffective treatment of inflammatory diseases (including cancers). Morespecifically, the present invention concerns the inhibition orneutralization of both IL-17 and IL-23 (via p19) with a singleantagonistic molecule or neutralizing entity.

The antagonistic molecule or neutralizing entity inhibits the activityof both IL-17 and IL-23 (via p19), and thus, inhibits the production,maintenance, and activity of new and existing IL-17 and IL-17-producingT cells (Th17). TH17 cells include IL-17A and IL-17F. The inventionfurther concerns the use of IL-17 and IL-23/p19 antagonists orneutralizing entities in the treatment of inflammatory diseasescharacterized by the presence of elevated levels of IL-17 and/or IL-23.The invention also concerns the use of IL-17 and IL-23/p19 antagonistsin the treatment of cancers characterized by the presence of elevatedlevels of IL-17 and/or IL-23.

Accordingly, the present invention is directed to antagonizing bothIL-17 and IL-23/p19, either singly or together. Since either IL-17,IL-23/p19, or IL-23/p40 intervention has been proposed as an effectivetherapy for several inflammatory diseases and various cancers, usingantagonists of the present invention, which may block, inhibit, reduce,antagonize or neutralize the activity of IL-17, IL-23, IL-23/p19 or bothIL-17A and IL-23 (via p19), will have advantages over therapies thattarget only one of these two cytokines. The invention further providesuses therefore in inflammatory disease and cancer, as well as relatedcompositions and methods.

A) Overview

Immune related and inflammatory diseases are the manifestation orconsequence of fairly complex, often multiple interconnected biologicalpathways which in normal physiology are critical to respond to insult orinjury, initiate repair from insult or injury, and mount innate andacquired defense against foreign organisms. Disease or pathology occurswhen these normal physiological pathways cause additional insult orinjury either as directly related to the intensity of the response, as aconsequence of abnormal regulation or excessive stimulation, as areaction to self, or as a combination of these.

Though the genesis of these diseases often involves multi-step pathwaysand often multiple different biological systems/pathways, interventionat critical points in one or more of these pathways can have anameliorative or therapeutic effect. Therapeutic intervention can occurby either antagonism of a detrimental process/pathway or stimulation ofa beneficial process/pathway.

Many immune related diseases are known and have been extensivelystudied. Such diseases include immune-mediated inflammatory diseases(such as rheumatoid arthritis, multiple sclerosis, demyelinatingdiseases, autoimmune ocular diseases, uveitis; scleritis, immunemediated renal disease, hepatobiliary diseases, inflammatory boweldisease (IBD), irritable bowel syndrome (IBS), psoriasis, and asthma),non-immune-mediated inflammatory diseases, infectious diseases,immunodeficiency diseases, neoplasia, etc.

T lymphocytes (T cells) are an important component of a mammalian immuneresponse. T cells recognize antigens which are associated with aself-molecule encoded by genes within the major histocompatibilitycomplex (MHC). The antigen may be displayed together with MHC moleculeson the surface of antigen-presenting cells, virus-infected cells, cancercells, grafts, etc. The T cell system eliminates these altered cellswhich pose a health threat to the host mammal. T cells include helper Tcells and cytotoxic T cells. Helper T cells proliferate extensivelyfollowing recognition of an antigen-MHC complex on an antigen presentingcell. Helper T cells also secrete a variety of cytokines, i.e.,lymphokines, which play a central role in the activation of B cells,cytotoxic T cells and a variety of other cells which participate in theimmune response.

A central event in both humoral and cell mediated immune responses isthe activation and clonal expansion of helper T cells. Helper T cellactivation is initiated by the interaction of the T cell receptor(TCR)-CD3 complex with an antigen-MHC on the surface of anantigen-presenting cell. This interaction mediates a cascade ofbiochemical events that induce the resting helper T cell to enter a cellcycle (the G0 to G1 transition) and results in the expression of a highaffinity receptor for IL-2 and sometimes IL-4. The activated T cellprogresses through the cycle proliferating and differentiating intomemory cells or effector cells.

In addition to the signals mediated through the TCR, activation of Tcells involves additional costimulation induced by cytokines released bythe antigen presenting cell or through interactions with membrane boundmolecules on the antigen presenting cell and the T cell. The cytokinesIL-1 and IL-6 have been shown to provide a costimulatory signal. Also,the interaction between the B7 molecule expressed on the surface of anantigen presenting cell and CD28 and CTLA-4 molecules expressed on the Tcell surface effect T cell activation. Activated T cells express anincreased number of cellular adhesion molecules, such as ICAM-1,integrins, VLA-4, LFA-1, CD56, etc.

T-cell proliferation in a mixed lymphocyte culture or mixed lymphocytereaction (MLR) is an established indication of the ability of a compoundto stimulate the immune system. In many immune responses, inflammatorycells infiltrate the site of injury or infection. The migrating cellsmay be neutrophilic, eosinophilic, monocytic or lymphocytic as can bedetermined by histologic examination of the affected tissues. CurrentProtocols in Immunology, ed. John E. Coligan, 1994, John Wiley & Sons,Inc.

Immune related diseases could be treated by suppressing the immuneresponse. Using the antagonists of the present invention (i.e.anti-IL-17 and/or anti-IL-23/p19 antibodies) that inhibit moleculeshaving immune stimulatory activity would be beneficial in the treatmentof immune-mediated and inflammatory diseases. Molecules which inhibitthe immune response can be utilized (proteins directly or via the use ofantibody agonists) to inhibit the immune response and thus ameliorateimmune related disease.

IL-17 has been identified as a cellular ortholog of a protein encoded bythe T lymphotropic Herpes virus Saimiri (HSV) [see, Rouvier et al., J.Immunol., 150(12): 5445-5456 (1993); Yao et al., J. Immunol.,122(12):5483-5486 (1995) and Yao et al., Immunity, 3(6):811-821 (1995)].Subsequent characterization has shown that this protein is a potentcytokine that acts to induce proinflammatory responses in a wide varietyof peripheral tissues. IL-17 is a disulfide-linked homodimeric cytokineof about 32 kDa which is synthesized and secreted primarily by CD4+activated memory T cells (reviewed in Fossiez et al., Int. Rev.Immunol., 16: 541-551 [1998]). Specifically, IL-17 is synthesized as aprecursor polypeptide of 155 amino acids with an N-terminal signalsequence of 19-23 residues and is secreted as a disulfide-linkedhomodimeric glycoprotein. IL-17 is disclosed in WO9518826 (1995),WO9715320 (1997) and WO9704097 (1997), as well as U.S. Pat. No.6,063,372.

Despite its restricted tissue distribution, IL-17 exhibits pleiotropicbiological activities on various types of cells. IL-17 has been found tostimulate the production of many cytokines. For example, it induces thesecretion of IL-6, IL-8, IL-12, leukemia inhibitory factor (LIF),prostaglandin E2, MCP-1 and G-CSF by adherent cells like fibroblasts,keratinocytes, epithelial and endothelial cells. IL-17 also has theability to induce ICAM-1 surface expression, proliferation of T cells,and growth and differentiation of CD34.sup.+ human progenitors intoneutrophils. IL-17 is also believed to play a key role in certain otherautoimmune disorders such as multiple sclerosis (Matusevicius et al.,Mult. Scler. 5:101 (1999); Park et al, Nat Immunol. 6:1133 (2005)).IL-17 has further been shown, by intracellular signalling, to stimulateCa.sup.2+ influx and a reduction in [cAMP], in human macrophages(Jovanovic et al, J. Immunol. 160:3513 (1998)). Fibroblasts treated withIL-17 induce the activation of NF-kappa.B, (Yao et al., Immunity, 3:811(1995), Jovanovic et al., supra), while macrophages treated with itactivate NF-kappa.B and mitogen-activated protein kinases (Shalom-Bareket al, J. Biol. Chem. 273:27467 (1998)).

Consistent with IL-17's wide-range of effects, the cell surface receptorfor IL-17 has been found to be widely expressed in many tissues and celltypes (Yao et al., Cytokine, 9:794 (1997)). While the amino acidsequence of the human IL-17 receptor (IL-17R) (866 amino acids) predictsa protein with a single transmembrane domain and a long, 525 amino acidintracellular domain, the receptor sequence is unique and is not similarto that of any of the receptors from the cytokine/growth factor receptorfamily. This coupled with the lack of similarity of IL-17 itself toother known proteins indicates that IL-17 and its receptor may be partof a novel family of signalling proteins and receptors. It has beendemonstrated that IL-17 activity is mediated through binding to itsunique cell surface receptor, wherein previous studies have shown thatcontacting T cells with a soluble form of the IL-17 receptor polypeptideinhibited T cell proliferation and IL-2 production induced by PHA,concanavalin A and anti-TCR monoclonal antibody (Yao et al, J. Immunol,155:5483 (1995)).

IL-17 and IL-23 appear to represent a unique signaling system within thecytokine network that will offer innovative approaches to themanipulation of immune and inflammatory responses.

As such, antagonists to IL-17 and IL-23 activity, such as theantagonists of the present invention (i.e. anti-IL-17 and/oranti-IL-23/p19 antibodies), are useful in therapeutic treatment ofinflammatory diseases, particularly as antagonists to both IL-17 andIL-23/p19, either singly or together in the treatment of inflammatorydiseases, particularly as antagonists to both IL-17 and IL-23/p19 in thetreatment of multiple sclerosis, inflammatory bowel disease (IBD),rheumatoid arthritis, psoriasis, and cancer. Moreover, antagonists toIL-17 and IL-23/p19 activity, such as the antagonists of the presentinvention (i.e. anti-IL-17 and/or anti-IL-23/p19 antibodies), are usefulin therapeutic treatment of other inflammatory diseases. Theseantagonists are capable of binding, blocking, inhibiting, reducing,antagonizing or neutralizing IL-17 and IL-23 (via p19) (eitherindividually or together) in the treatment of atopic and contactdermatitis, colitis, endotoxemia, arthritis, rheumatoid arthritis,psoriatic arthritis, autoimmune ocular diseases (uveitis, scleritis),adult respiratory disease (ARD), demyelinating diseases, septic shock,multiple organ failure, inflammatory lung injury such as asthma, chronicobstructive pulmonary disease (COPD), airway hyper-responsiveness,chronic bronchitis, allergic asthma, psoriasis, eczema, EBS andinflammatory bowel disease (IBD) such as ulcerative colitis and Crohn'sdisease, diabetes, Helicobacter pylori infection, intraabdominaladhesions and/or abscesses as results of peritoneal inflammation (i.e.from infection, injury, etc.), systemic lupus erythematosus (SLE),multiple sclerosis, systemic sclerosis, nephrotic syndrome, organallograft rejection, graft vs. host disease (GVHD), kidney, lung, heart,etc. transplant rejection, streptococcal cell wall (SCW)-inducedarthritis, osteoarthritis, gingivitis/periodontitis, herpetic stromalkeratitis, restenosis, Kawasaki disease, and cancers/neoplastic diseasesthat are characterized by IL-17 and/or IL-23 expression, including butnot limited to prostate, renal, colon, ovarian and cervical cancer, andleukemias (Tartour et al, Cancer Res. 59:3698 (1999); Kato et al,Biochem. Biophys. Res. Commun. 282:735 (2001); Steiner et al, Prostate.56:171 (2003); Langowksi et al, Nature 442: 461, 2006).

Amongst other inventions, the present invention provides novelantagonists of IL-17 and IL-23/p19 and their uses in the treatment ofinflammatory diseases and autoimmune diseases. The IL-17 and IL-23/p19antagonists of the present invention, including the neutralizinganti-IL-17 and IL-23/p19 antibodies of the present invention, can beused to block, inhibit, reduce, antagonize or neutralize the activity ofeither IL-17 or IL-23 (via p19), or both IL-17 and IL-23 (via p19) inthe treatment of inflammation and inflammatory diseases such as multiplesclerosis, cancer (as characterized by the expression of IL-17 and/orIL-23), psoriasis, psoriatic arthritis, rheumatoid arthritis, autoimmuneocular diseases, endotoxemia, IBS, and inflammatory bowel disease (IBD),colitis, asthma, allograft rejection, immune mediated renal diseases,hepatobiliary diseases, atherosclerosis, promotion of tumor growth, ordegenerative joint disease and other inflammatory conditions disclosedherein.

The present invention provides isolated polypeptides that bind IL-17(e.g., human IL-17 polypeptide sequence as shown in SEQ ID NO:2). Thepresent invention also provides isolated polypeptides as disclosed abovethat bind IL-23 (e.g., human IL-23 polypeptide sequence as shown in SEQID NO:6). More specifically, the present invention provides polypeptidesthat bind to the p19 subunit of IL-23 (e.g. human p19 polypeptidesequence as shown in SEQ ID NO:4).

The present invention also provides isolated polypeptides and epitopescomprising at least 15 contiguous amino acid residues of an amino acidsequence of SEQ ID NO:2 or 4. Illustrative polypeptides includepolypeptides that either comprise, or consist of SEQ ID NO:2 or 4, anantigenic epitope thereof. Moreover, the present invention also providesisolated polypeptides as disclosed above that bind to, block, inhibit,reduce, antagonize or neutralize the activity of IL-17 or IL-23.

Preferred embodiments of the invention include binding peptides,antibodies, and any fragments or permutations thereof that bind to IL-17or IL-23/p19 (herein referred to interchangeably as “IL-17/IL-23antagonists”, “IL-17 antagonists”, “IL-23 antagonists”, “p19antagonists”, “IL-17/IL-23 antibodies”, “IL-17/p19 antibodies”, “IL-17antibodies”, “IL-23 antibodies”, “p19 antibodies”, “IL-17/IL-23/p19antibodies”, IL-17A neutralizing entities, IL-23p19 neutralizingentities, etc.). Specifically, such binding peptides or antibodies arecapable of specifically binding to both human IL-17 and IL-23 (via p19)and/or are capable of modulating biological activities associated witheither or both IL-17 and IL-23, and thus are useful in the treatment ofvarious diseases and pathological conditions such as inflammation andimmune-related diseases.

Thus, the present invention provides antibodies and antibody fragmentsthat specifically bind with IL-17 and/or IL-23 (via p19). Exemplaryantibodies include neutralizing antibodies, polyclonal antibodies,murine monoclonal antibodies, chimeric antibodies, humanized antibodiesderived from murine monoclonal antibodies, and human monoclonalantibodies. Illustrative antibody fragments include F(ab′)₂, F(ab)₂,Fab′, Fab, Fv, scFv, and minimal recognition units. Neutralizingantibodies preferably bind IL-17 or IL-23/p19 such that the interactionof IL-17 and IL-23 with their respective receptors (i.e. IL-17RA orIL-17RC for IL-17; IL-12b1 and IL-23R for IL-23) is blocked, inhibited,reduced, antagonized or neutralized. That is, the neutralizing IL-17 andIL-23/p19 antibodies of the present invention can either bind, block,inhibit, reduce, antagonize or neutralize each of IL-17 or IL-23 singly,or bind, block, inhibit, reduce, antagonize or neutralize IL-17 andIL-23 together. The present invention further includes compositionscomprising a carrier and a peptide, polypeptide, or antibody describedherein.

The present invention further includes pharmaceutical compositions,comprising a pharmaceutically acceptable carrier and a polypeptide orantibody described herein.

The present invention also contemplates anti-idiotype antibodies, oranti-idiotype antibody fragments, that specifically bind an antibody orantibody fragment that specifically binds a polypeptide comprising theamino acid sequence of SEQ ID NO:2, 4 or any fragment thereof. Anexemplary anti-idiotype antibody binds with an antibody thatspecifically binds a polypeptide consisting of SEQ ID NOS:2 or 4.

The present invention also provides fusion proteins, comprising anantagonist of the present invention and an immunoglobulin moiety. Insuch fusion proteins, the immunoglobulin moiety may be an immunoglobulinheavy chain constant region, such as a human F_(C) fragment. The presentinvention further includes isolated nucleic acid molecules that encodesuch fusion proteins.

In a particular embodiment, the present invention provides bispecificantibodies or binding proteins that bind both IL-17 and IL-23.Bispecific antibodies (BsAbs) are antibodies that have two differentantigen binding sites, such that the antibody specifically binds to twodifferent antigens. Antibodies having higher valencies (i.e., theability to bind to more than two antigens) can also be prepared; theyare referred to as multispecific antibodies.

The bispecific antibody can be a monoclonal antibody (MAb) with respectto each target. In particular embodiments, the antibody is chimeric, orhumanized, or fully human. Fully human antibodies may be generated byprocedures that involve immunizing transgenic mice, wherein humanimmunoglobulin genes have been introduced into the mice, as discussedbelow. Bispecific antibodies of the invention, which bind IL-17 andIL-23 (via p19), are referred to herein as bispecific IL-17/IL-23antibodies or bispecific IL-17/p19 MAbs.

In yet other particular embodiments, there is provided the hybridomacell line which produces monoclonal antibodies of the present invention.In another embodiment, the IL-17/IL-23 antibodies are linked to one ormore non-proteinaceous polymers selected from the group consisting ofpolyethylene glycol, polypropylene glycol, and polyoxyalkylene, or to acytotoxic agent or enzyme, or to a radioisotope, fluorescent compound orchemiluminescent compound.

Typical methods of the invention include methods to treat pathologicalconditions or diseases in mammals associated with or resulting fromincreased or enhanced IL-17 and/or IL-23 expression and/or activity. Inthe methods of treatment, the antibodies of the present invention may beadministered which preferably block or reduce the respective receptorbinding or activation to their receptor(s). Optionally, the antibodiesemployed in the methods will be capable of blocking or neutralizing theactivity of both IL-17 and IL-23(p19), e.g., a dual antagonist whichblocks or neutralizes activity of IL-17A or IL-23. The methodscontemplate the use of a single bispecific binding peptide or antibodyor a combination of two or more antibodies (each of which specificallybinds to either IL-17 or IL-23 via p19).

The invention also provides compositions which comprise IL-17/IL-23antibodies. Optionally, the compositions of the invention will includepharmaceutically acceptable carriers or diluents. Preferably, thecompositions will include one or more IL-17/IL-23 antibodies in anamount which is therapeutically effective to treat a pathologicalcondition or disease.

As such, the present invention concerns compositions and methods usefulfor the diagnosis and treatment of inflammation or immune-relateddisease in mammals, including humans. The present invention is based onthe identification of the synergistic effect of neutralizing both IL-17and IL-23 as compared with neutralization of one alone. Immune relateddiseases can be treated by suppressing or enhancing the immune response.Antibodies that enhance the immune response stimulate or potentiate theimmune response to an antigen. Antibodies which stimulate the immuneresponse can be used therapeutically where enhancement of the immuneresponse would be beneficial. Alternatively, antibodies that suppressthe immune response attenuate or reduce the immune response to anantigen (e.g., neutralizing antibodies) can be used therapeuticallywhere attenuation of the immune response would be beneficial (e.g.,inflammation).

Accordingly, antagonists of the present invention (i.e. antibodies orbinding peptides that bind IL-17 and IL-23 either singly or together)are also useful to prepare medicines and medicaments for the treatmentof immune-related and inflammatory diseases, including for example,systemic lupus erythematosis, arthritis, rheumatoid arthritis,osteoarthritis, psoriasis, demyelinating diseases of the central andperipheral nervous systems such as multiple sclerosis, idiopathicdemyelinating polyneuropathy or Guillain-Barre syndrome, inflammatorybowel disease, colitis, ulcerative colitis, Crohn's disease,gluten-sensitive enteropathy, autoimmune ocular diseases, cancer,neoplastic diseases, and angiogenesis.

In a specific aspect, such medicines and medicaments comprise atherapeutically effective amount of an IL-17/IL-23 antibody with apharmaceutically acceptable carrier. Preferably, the admixture issterile.

In a further embodiment, the invention concerns a method of identifyingantagonist antibodies of IL-17 and IL-23/p19, said method comprisingcontacting both IL-17 and p19 with a candidate molecule and monitoring abiological activity mediated by IL-17 and/or IL-23. In anotherembodiment, the invention concerns a composition of matter comprising anIL-17/IL-23 antagonist antibody which binds both IL-17 and IL-23 inadmixture with a carrier or excipient. In one aspect, the compositioncomprises a therapeutically effective amount of the IL-17/IL-23antibody. The composition is useful for: (a) reducing infiltration ofinflammatory cells into a tissue of a mammal in need thereof, (b)inhibiting or reducing an immune response in a mammal in need thereof,(c) decreasing the proliferation of T-lymphocytes in a mammal in needthereof in response to an antigen, (d) inhibiting the activity ofT-Lymphocytes, (e) decreasing the vascular permeability, or (f) reducingsystemic and/or local concentrations of IL-17 and/or IL-23.

In another embodiment, the invention concerns a method of treating animmune related disorder in a mammal in need thereof, comprisingadministering to the mammal a therapeutically effective amount of anIL-17/IL-23 antagonist.

Optionally, the antibody is a monoclonal antibody, humanized antibody,antibody fragment or single-chain antibody. In another embodiment, theinvention provides an antibody which specifically binds to both IL-17and IL-23. The antibody may be labeled and may be immobilized on a solidsupport. In a further aspect, the antibody is an antibody fragment, amonoclonal antibody, a single-chain antibody, or an anti-idiotypicantibody.

In still another embodiment, the invention concerns an isolatedpolynucleotide that encodes a polypeptide of the present invention,wherein said polypeptide is capable of binding to both IL-17 and IL-23.

In still another embodiment, the invention concerns an isolatedpolypeptide of the present invention, wherein said polypeptide iscapable of binding to both IL-17 and IL-23.

In yet another embodiment, the invention concerns a method forinhibiting IL-17 production and/or maintenance by treating the T cellswith an antagonist of IL-23/p19 (IL-23).

In another aspect, the invention concerns a method for the treatment ofan inflammatory disease characterized by elevated expression of IL-17and IL-23 in a mammalian subject, comprising administering to thesubject an effective amount of an antagonist of IL-17 and IL-23

In yet another aspect, the invention concerns a method for identifyingan anti-inflammatory agent comprising the steps of (a) incubating aculture of T cells with IL-23, in the presence and absence of acandidate molecule; (b) monitoring the level of IL-17 in the culture;and (c) identifying the candidate molecule as an anti-inflammatory agentif the level of IL-17 is lower in the presence than in the absence ofsuch candidate molecule.

Processes for producing the same are also herein described, whereinthose processes comprise culturing a host cell comprising a vector whichcomprises the appropriate encoding nucleic acid molecule underconditions suitable for expression of said antibody and recovering saidantibody from the cell culture.

In yet another embodiment, the present invention provides a compositioncomprising an IL-17/IL-23 antibody in admixture with a pharmaceuticallyacceptable carrier. In one aspect, the composition comprises atherapeutically effective amount of the antibody. Preferably, thecomposition is sterile. The composition may be administered in the formof a liquid pharmaceutical formulation, which may be preserved toachieve extended storage stability. Alternatively, the antibody is amonoclonal antibody, an antibody fragment, a humanized antibody, or asingle-chain antibody.

In a further embodiment, the invention concerns an article ofmanufacture, comprising: (a) a composition of matter comprising anIL-17/IL-23 antibody, or an antibody that specifically binds to bothIL-17 and IL-23 (via p19); (b) a container containing said composition;and (c) a label affixed to said container, or a package insert includedin said container referring to the use of said IL-17/IL-23 antibodythereof in the treatment of an immune related disease. The compositionmay comprise a therapeutically effective amount of the IL-17/IL-23antibody.

In yet another embodiment, the present invention concerns a method ofdiagnosing an immune related disease in a mammal, comprising detectingthe level of expression of a gene encoding either or both IL-17 and/orIL-23 (a) in a test sample of tissue cells obtained from the mammal, and(b) in a control sample of known normal tissue cells of the same celltype, wherein a higher or lower expression level in the test sample ascompared to the control sample indicates the presence of immune relateddisease in the mammal from which the test tissue cells were obtained.

In another embodiment, the present invention concerns a method ofdiagnosing an immune disease in a mammal, comprising (a) contacting anIL-17/IL-23 antibody with a test sample of tissue cells obtained fromthe mammal, and (b) detecting the formation of a complex between theantibody and either or both IL-17 and IL-23 in the test sample; whereinthe formation of said complex is indicative of the presence or absenceof said disease. The detection may be qualitative or quantitative, andmay be performed in comparison with monitoring the complex formation ina control sample of known normal tissue cells of the same cell type. Alarger quantity of complexes formed in the test sample indicates thepresence or absence of an immune disease in the mammal from which thetest tissue cells were obtained. The antibody preferably carries adetectable label. Complex formation can be monitored, for example, bylight microscopy, flow cytometry, fluorimetry, or other techniques knownin the art. The test sample is usually obtained from an individualsuspected of having a deficiency or abnormality of the immune system.

In another embodiment, the invention provides a method of diagnosing animmune-related disease in a mammal which comprises detecting thepresence or absence of both IL-17 and IL-23 in a test sample of tissuecells obtained from said mammal, wherein the presence or absence of bothIL-17 and IL-23 in said test sample is indicative of the presence of animmune-related disease in said mammal.

In a still further embodiment, the invention provides a method ofdecreasing the activity of T-lymphocytes in a mammal comprisingadministering to said mammal an IL-17/IL-23 antagonist, such as anIL-17/IL-23p19 antibody, wherein the activity of T-lymphocytes in themammal is decreased.

In a still further embodiment, the invention provides a method ofdecreasing the proliferation, of T-lymphocytes in a mammal comprisingadministering to said mammal (a) an IL-17/IL-23 antagonist, such as anIL-17/IL-23p19 antibody, wherein the proliferation of T-lymphocytes inthe mammal is decreased.

The invention also provides articles of manufacture and kits whichinclude one or more IL-17, IL-23 or IL-17/IL-23 antibodies.

These and other aspects of the invention will become evident uponreference to the following detailed description. In addition, variousreferences are identified below and are incorporated by reference intheir entirety.

B) Definitions

In the description that follows, a number of terms are used extensively.The following definitions are provided to facilitate understanding ofthe invention.

“Antibodies” (Abs) and “immunoglobulins” (Igs) are glycoproteins havingthe same structural characteristics. While antibodies exhibit bindingspecificity to a specific antigen, immunoglobulins include bothantibodies and other antibody-like molecules that lack antigenspecificity. Polypeptides of the latter kind are, for example, producedat low levels by the lymph system and at increased levels by myelomas.Thus, as used herein, the term “antibody” or “antibody peptide(s)”refers to an intact antibody, or a binding fragment thereof thatcompetes with the intact antibody for specific binding and includeschimeric, humanized, fully human, and bispecific antibodies. In certainembodiments, binding fragments are produced by recombinant DNAtechniques. In additional embodiments, binding fragments are produced byenzymatic or chemical cleavage of intact antibodies. Binding fragmentsinclude, but are not limited to, Fab, Fab′, F(ab).sub.2, F(ab′).sub.2,Fv, and single-chain antibodies. “Native antibodies and immunoglobulins”are usually heterotetrameric glycoproteins of about 150,000 daltons,composed of two identical light (L) chains and two identical heavy (H)chains. Each light chain is linked to a heavy chain by one covalentdisulfide bond, while the number of disulfide-linkages varies betweenthe heavy chains of different immunoglobulin isotypes. Each heavy andlight chain also has regularly spaced intrachain disulfide bridges. Eachheavy chain has at one end a variable domain (VH) followed by a numberof constant domains. Each light chain has a variable domain at one end(VL) and a constant domain at its other end; the constant domain of thelight chain is aligned with the first constant domain of the heavychain, and the light chain variable domain is aligned with the variabledomain of the heavy chain. Particular amino acid residues are believedto form an interface between the light- and heavy-chain variable domains(Clothia et al., J. Mol. Biol. 186:651 (1985); Novotny and Haber, Proc.Natl. Acad. Sci. U.S.A. 82:4592 (1985)).

The term “isolated antibody” as used herein refers to an antibody thathas been identified and separated and/or recovered from a component ofits natural environment. Contaminant components of its naturalenvironment are materials which would interfere with diagnostic ortherapeutic uses for the antibody, and may include enzymes, hormones,and other proteinaceous or nonproteinaceous solutes. In preferredembodiments, the antibody will be purified (1) to greater than 95% byweight of antibody as determined by the Lowry method, and mostpreferably more than 99% by weight, (2) to a degree sufficient to obtainat least 15 residues of N-terminal or internal amino acid sequence byuse of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGEunder reducing or nonreducing conditions using Coomassie blue or,preferably, silver stain. Isolated antibody includes the antibody insitu within recombinant cells since at least one component of theantibody's natural environment will not be present. Ordinarily, however,isolated antibody will be prepared by at least one purification step.

A “variant” anti-IL-17 and IL-23 antibody, and/or IL-17/IL-23 antibody,refers herein to a molecule which differs in amino acid sequence from a“parent” antibody amino acid sequence by virtue of addition, deletionand/or substitution of one or more amino acid residue(s) in the parentantibody sequence. In the preferred embodiment, the variant comprisesone or more amino acid substitution(s) in one or more hypervariableregion(s) of the parent antibody. For example, the variant may compriseat least one, e.g. from about one to about ten, and preferably fromabout two to about five, substitutions in one or more hypervariableregions of the parent antibody. Ordinarily, the variant will have anamino acid sequence having at least 75% amino acid sequence identitywith the parent antibody heavy or light chain variable domain sequences,more preferably at least 80%, more preferably at least 85%, morepreferably at least 90%, and most preferably at least 95%. Identity orhomology with respect to this sequence is defined herein as thepercentage of amino acid residues in the candidate sequence that areidentical with the parent antibody residues, after aligning thesequences and introducing gaps, if necessary, to achieve the maximumpercent sequence identity. None of N-terminal, C-terminal, or internalextensions, deletions, or insertions into the antibody sequence shall beconstrued as affecting sequence identity or homology. The variantretains the ability to bind human IL-17 and/or IL-23 (via p19) andpreferably has properties which are superior to those of the parentantibody. For example, the variant may have a stronger binding affinity,enhanced ability to inhibit IL-17A and/or IL-23-induced inflammation. Toanalyze such properties, one should compare a Fab form of the variant toa Fab form of the parent antibody or a full length form of the variantto a full length form of the parent antibody, for example, since it hasbeen found that the format of the anti-IL-17/IL-23 antibody impacts itsactivity in the biological activity assays disclosed herein. The variantantibody of particular interest herein is one which displays at leastabout 10-fold, preferably at least about 20-fold, and most preferably atleast about 50-fold, enhancement in biological activity when compared tothe parent antibody.

The term “parent antibody” as used herein refers to an antibody which isencoded by an amino acid sequence used for the preparation of thevariant. Preferably, the parent antibody has a human framework regionand, if present, has human antibody constant region(s). For example, theparent antibody may be a humanized or human antibody.

The term “agonist” refers to any compound including a protein,polypeptide, peptide, antibody, antibody fragment, large molecule, orsmall molecule (less than 10 kD), that increases the activity,activation or function of another molecule.

The term “antagonist” refers to any compound including a protein,polypeptide, peptide, antibody, antibody fragment, large molecule, orsmall molecule (less than 10 kD), that decreases the activity,activation or function of another molecule.

The term “bind(ing) of a polypeptide of the invention to a ligand”includes, but is not limited to, the binding of a ligand polypeptide ofthe present invention to a receptor; the binding of a receptorpolypeptide of the present invention to a ligand; the binding of anantibody of the present invention to an antigen or epitope; the bindingof an antigen or epitope of the present invention to an antibody; thebinding of an antibody of the present invention to an anti-idiotypicantibody; the binding of an anti-idiotypic antibody of the presentinvention to a ligand; the binding of an anti-idiotypic antibody of thepresent invention to a receptor; the binding of an anti-anti-idiotypicantibody of the present invention to a ligand, receptor or antibody,etc.

A “bivalent antibody” other than a “multispecific” or “multifunctional”antibody, in certain embodiments, is understood to comprise bindingsites having identical antigenic specificity.

A “bispecific” or “bifunctional” antibody is a hybrid antibody havingtwo different heavy/light chain pairs and two different binding sites.Bispecific antibodies may be produced by a variety of methods including,but not limited to, fusion of hybridomas or linking of Fab′ fragments.See, e.g., Songsivilai & Lachmann (1990), Clin. Exp. Immunol.79:315-321; Kostelny et al. (1992), J. Immunol. 148:1547-1553.

The term “chimeric antibody” or “chimeric antibodies” refers toantibodies whose light and heavy chain genes have been constructed,typically by genetic engineering, from immunoglobulin variable andconstant region genes belonging to different species. For example, thevariable segments of the genes from a mouse monoclonal antibody may bejoined to human constant segments, such as gamma 1 and gamma 3. Atypical therapeutic chimeric antibody is thus a hybrid protein composedof the variable or antigen-binding domain from a mouse antibody and theconstant domain from a human antibody, although other mammalian speciesmay be used. Specifically, a chimeric antibody is produced byrecombinant DNA technology in which all or part of the hinge andconstant regions of an immunoglobulin light chain, heavy chain, or both,have been substituted for the corresponding regions from anotheranimal's immunoglobulin light chain or heavy chain. In this way, theantigen-binding portion of the parent monoclonal antibody is graftedonto the backbone of another species' antibody. One approach, describedin EP 0239400 to Winter et al. describes the substitution of onespecies' complementarity determining regions (CDRs) for those of anotherspecies, such as substituting the CDRs from human heavy and light chainimmunoglobulin variable region domains with CDRs from mouse variableregion domains. These altered antibodies may subsequently be combinedwith human immunoglobulin constant regions to form antibodies that arehuman except for the substituted murine CDRs which are specific for theantigen. Methods for grafting CDR regions of antibodies may be found,for example in Riechmann et al. (1988) Nature 332:323-327 and Verhoeyenet al. (1988) Science 239:1534-1536.

The term “effective neutralizing titer” as used herein refers to theamount of antibody which corresponds to the amount present in the serumof animals (human or cotton rat) that has been shown to be eitherclinically efficacious (in humans) or to reduce virus by 99% in, forexample, cotton rats. The 99% reduction is defined by a specificchallenge of, e.g., 10³ pfu, 10⁴ pfu, 10⁵ pfu, 10⁶ pfu, 10⁷ pfu, 10⁸pfu, or 10⁹ pfu) of RSV.

As used herein, the term “epitope” refers to the portion of an antigento which a monoclonal antibody specifically binds. Thus, the term“epitope” includes any protein determinant capable of specific bindingto an immunoglobulin or T-cell receptor. Epitopic determinants usuallyconsist of chemically active surface groupings of molecules such asamino acids or sugar side chains and usually have specific threedimensional structural characteristics, as well as specific chargecharacteristics. More specifically, the term “IL-17 epitope”, “IL-23epitope” and/or “IL-23/p19 epitope” as used herein refers to a portionof the corresponding polypeptide having antigenic or immunogenicactivity in an animal, preferably in a mammal, and most preferably in amouse or a human. An epitope having immunogenic activity is a portion ofan IL-17 and/or IL-23/p19 polypeptide that elicits an antibody responsein an animal. An epitope having antigenic activity is a portion of anIL-17 and/or IL-23/p19 polypeptide to which an antibodyimmunospecifically binds as determined by any method well known in theart, for example, by immunoassays. Antigenic epitopes need notnecessarily be immunogenic. Such epitopes can be linear in nature or canbe a discontinuous epitope. Thus, as used herein, the term“conformational epitope” refers to a discontinuous epitope formed by aspatial relationship between amino acids of an antigen other than anunbroken series of amino acids.

The term “epitope tagged” when used herein refers to the anti-IL-17and/or IL-23/p19 antibody fused to an “epitope tag”. The epitope tagpolypeptide has enough residues to provide an epitope against which anantibody can be made, yet is short enough such that it does notinterfere with activity of antibodies of the present invention. Theepitope tag preferably is sufficiently unique so that the antibodythereagainst does not substantially cross-react with other epitopes.Suitable tag polypeptides generally have at least 6 amino-acid residuesand usually between about 8-50 amino acid residues (preferably betweenabout 9-30 residues). Examples include the flu HA tag polypeptide andits antibody 12CA5 (Field et al. Mol. Cell. Biol. 8:2159-2165 (1988));the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereto(Evan et al., Mol. Cell. Biol. 5(12):3610-3616 (1985)); and the HerpesSimplex virus glycoprotein D (gD) tag and its antibody (Paborsky et al.,Protein Engineering 3(6):547-553 (1990)). In certain embodiments, theepitope tag is a “salvage receptor binding epitope”. As used herein, theterm “salvage receptor binding epitope” refers to an epitope of the Fcregion of an IgG molecule (e.g., IgG.sub.1, IgG.sub.2, IgG.sub.3, orIgG.sub.4) that is responsible for increasing the in vivo serumhalf-life of the IgG molecule.

The term “fragment” as used herein refers to a peptide or polypeptidecomprising an amino acid sequence of at least 5 contiguous amino acidresidues, at least 10 contiguous amino acid residues, at least 15contiguous amino acid residues, at least 20 contiguous amino acidresidues, at least 25 contiguous amino acid residues, at least 40contiguous amino acid residues, at least 50 contiguous amino acidresidues, at least 60 contiguous amino residues, at least 70 contiguousamino acid residues, at least contiguous 80 amino acid residues, atleast contiguous 90 amino acid residues, at least contiguous 100 aminoacid residues, at least contiguous 125 amino acid residues, at least 150contiguous amino acid residues, at least contiguous 175 amino acidresidues, at least contiguous 200 amino acid residues, or at leastcontiguous 250 amino acid residues of the amino acid sequence of a IL-17or IL-23/p19 polypeptide or an antibody that immunospecifically binds toa either IL-17 or IL-23 (via p19) or both IL-17 and IL-23/p19polypeptide.

As used herein, the term “immunoglobulin” refers to a protein consistingof one or more polypeptides substantially encoded by immunoglobulingenes. One form of immunoglobulin constitutes the basic structural unitof an antibody. This form is a tetramer and consists of two identicalpairs of immunoglobulin chains, each pair having one light and one heavychain. In each pair, the light and heavy chain variable regions aretogether responsible for binding to an antigen, and the constant regionsare responsible for the antibody effector functions.

Full-length immunoglobulin “light chains” (about 25 Kd or 214 aminoacids) are encoded by a variable region gene at the NH2-terminus (about110 amino acids) and a kappa or lambda constant region gene at theCOOH-terminus Full-length immunoglobulin “heavy chains” (about 50 Kd or446 amino acids), are similarly encoded by a variable region gene (about116 amino acids) and one of the other aforementioned constant regiongenes (about 330 amino acids). Heavy chains are classified as gamma, mu,alpha, delta, or epsilon, and define the antibody's isotype as IgG, IgM,IgA, IgD and IgE, respectively. Within light and heavy chains, thevariable and constant regions are joined by a “J” region of about 12 ormore amino acids, with the heavy chain also including a “D” region ofabout 10 more amino acids. (See generally, Fundamental Immunology (Paul,W., ed., 2nd ed. Raven Press, N.Y., 1989), Ch. 7 (incorporated byreference in its entirety for all purposes).

An immunoglobulin light or heavy chain variable region consists of a“framework” region interrupted by three hypervariable regions. Thus, theterm “hypervariable region” refers to the amino acid residues of anantibody which are responsible for antigen binding. The hypervariableregion comprises amino acid residues from a “Complementarity DeterminingRegion” or “CDR” (i.e., residues 24-34 (L1), 50-56 (L2) and 89-97 (L3)in the light chain variable domain and 31-35 (H1), 50-65 (H2) and 95-102(H3) in the heavy chain variable domain (Kabat et al., Sequences ofProteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991)) and/or thoseresidues from a “hypervariable loop” (i.e., residues 26-32 (L1), 50-52(L2) and 91-96 (L3) in the light chain variable domain and 26-32 (H1),53-55 (H2) and 96-101 (H3) in the heavy chain variable domain; Chothiaand Lesk, 1987, J. Mol. Biol. 196: 901-917) (both of which areincorporated herein by reference). “Framework Region” or “FR” residuesare those variable domain residues other than the hypervariable regionresidues as herein defined. The sequences of the framework regions ofdifferent light or heavy chains are relatively conserved within aspecies. Thus, a “human framework region” is a framework region that issubstantially identical (about 85% or more, usually 90-95% or more) tothe framework region of a naturally occurring human immunoglobulin. Theframework region of an antibody, that is the combined framework regionsof the constituent light and heavy chains, serves to position and alignthe CDR's. The CDR's are primarily responsible for binding to an epitopeof an antigen.

Accordingly, the term “humanized” immunoglobulin refers to animmunoglobulin comprising a human framework region and one or more CDR'sfrom a non-human (usually a mouse or rat) immunoglobulin. The non-humanimmunoglobulin providing the CDR's is called the “donor” and the humanimmunoglobulin providing the framework is called the “acceptor”.Constant regions need not be present, but if they are, they must besubstantially identical to human immunoglobulin constant regions, i.e.,at least about 85-90%, preferably about 95% or more identical. Hence,all parts of a humanized immunoglobulin, except possibly the CDR's, aresubstantially identical to corresponding parts of natural humanimmunoglobulin sequences. A “humanized antibody” is an antibodycomprising a humanized light chain and a humanized heavy chainimmunoglobulin. For example, a humanized antibody would not encompass atypical chimeric antibody as defined above, e.g., because the entirevariable region of a chimeric antibody is non-human.

As used herein, the term “human antibody” includes and antibody that hasan amino acid sequence of a human immunoglobulin and includes antibodiesisolated from human immunoglobulin libraries or from animals transgenicfor one or more human immunoglobulin and that do not express endogenousimmunoglobulins, as described, for example, by Kucherlapati et al. inU.S. Pat. No. 5,939,598.

The term “genetically altered antibodies” means antibodies wherein theamino acid sequence has been varied from that of a native antibody.Because of the relevance of recombinant DNA techniques in the generationof antibodies, one need not be confined to the sequences of amino acidsfound in natural antibodies; antibodies can be redesigned to obtaindesired characteristics. The possible variations are many and range fromthe changing of just one or a few amino acids to the complete redesignof, for example, the variable or constant region. Changes in theconstant region will, in general, be made in order to improve or altercharacteristics, such as complement fixation, interaction with membranesand other effector functions. Changes in the variable region will bemade in order to improve the antigen binding characteristics.

In addition to antibodies, immunoglobulins may exist in a variety ofother forms including, for example, single-chain or Fv, Fab, and(Fab′).sub.2, as well as diabodies, linear antibodies, multivalent ormultispecific hybrid antibodies (as described above and in detail in:Lanzavecchia et al., Eur. J. Immunol. 17, 105 (1987)) and in singlechains (e.g., Huston et al., Proc. Natl. Acad. Sci. U.S.A., 85,5879-5883 (1988) and Bird et al., Science, 242, 423-426 (1988), whichare incorporated herein by reference). (See, generally, Hood et al.,“Immunology”, Benjamin, N.Y., 2nd ed. (1984), and Hunkapiller and Hood,Nature, 323, 15-16 (1986), which are incorporated herein by reference).

As used herein, the terms “single-chain Fv,” “single-chain antibodies,”“Fv” or “scFv” refer to antibody fragments that comprises the variableregions from both the heavy and light chains, but lacks the constantregions, but within a single polypeptide chain. Generally, asingle-chain antibody further comprises a polypeptide linker between theVH and VL domains which enables it to form the desired structure whichwould allow for antigen binding. Single chain antibodies are discussedin detail by Pluckthun in The Pharmacology of Monoclonal Antibodies,vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp.269-315 (1994). Various methods of generating single chain antibodiesare known, including those described in U.S. Pat. Nos. 4,694,778 and5,260,203; International Patent Application Publication No. WO 88/01649;Bird (1988) Science 242:423-442; Huston et al. (1988) Proc. Natl. Acad.Sci. USA 85:5879-5883; Ward et al. (1989) Nature 334:54454; Skerra etal. (1988) Science 242:1038-1041, the disclosures of which areincorporated by reference for any purpose. In specific embodiments,single-chain antibodies can also be bi-specific and/or humanized.

A “Fab fragment” is comprised of one light chain and the C.sub.H1 andvariable regions of one heavy chain. The heavy chain of a Fab moleculecannot form a disulfide bond with another heavy chain molecule.

A “Fab′ fragment” contains one light chain and one heavy chain thatcontains more of the constant region, between the C.sub.H1 and C.sub.H2domains, such that an interchain disulfide bond can be formed betweentwo heavy chains to form a F(ab′).sub.2 molecule.

A “F(ab′).sub.2 fragment” contains two light chains and two heavy chainscontaining a portion of the constant region between the C.sub.H1 andC.sub.H2 domains, such that an interchain disulfide bond is formedbetween two heavy chains.

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy chain variabledomain (V.sub.H) connected to a light chain variable domain (V.sub.L) inthe same polypeptide chain (V.sub.H-V.sub.L). By using a linker that istoo short to allow pairing between the two domains on the same chain,the domains are forced to pair with the complementary domains of anotherchain and create two antigen-binding sites. Diabodies are described morefully in, for example, EP 404,097; WO 93/11161; and Hollinger et al.,Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993).

The term “linear antibodies” refers to the antibodies described inZapata et al. Protein Eng. 8(10):1057-1062 (1995). Briefly, theseantibodies comprise a pair of tandem Fd segments(V_(H)-C_(H1)-V_(H)-C_(H1)) which form a pair of antigen bindingregions. Linear antibodies can be bispecific or monospecific.

The term “immunologically functional immunoglobulin fragment” as usedherein refers to a polypeptide fragment that contains at least thevariable domains of the immunoglobulin heavy and light chains. Animmunologically functional immunoglobulin fragment of the invention iscapable of binding to a ligand, preventing binding of the ligand to itsreceptor, interrupting the biological response resulting from ligandbinding to the receptor, or any combination thereof. Preferably, animmunologically functional immunoglobulin fragment of the inventionbinds specifically to either IL-17 or IL-23/p19 or to both IL-17 andIL-23/p19.

The term “monoclonal antibody” as used herein is not limited toantibodies produced through hybridoma technology. The term “monoclonalantibody” refers to an antibody that is derived from a single clone,including any eukaryotic, prokaryotic, or phage clone, and not themethod by which it is produced.

As used herein, “nucleic acid” or “nucleic acid molecule” refers topolynucleotides, such as deoxyribonucleic acid (DNA) or ribonucleic acid(RNA), oligonucleotides, fragments generated by the polymerase chainreaction (PCR), and fragments generated by any of ligation, scission,endonuclease action, and exonuclease action. Nucleic acid molecules canbe composed of monomers that are naturally-occurring nucleotides (suchas DNA and RNA), or analogs of naturally-occurring nucleotides (e.g.,α-enantiomeric forms of naturally-occurring nucleotides), or acombination of both. Modified nucleotides can have alterations in sugarmoieties and/or in pyrimidine or purine base moieties. Sugarmodifications include, for example, replacement of one or more hydroxylgroups with halogens, alkyl groups, amines, and azido groups, or sugarscan be functionalized as ethers or esters. Moreover, the entire sugarmoiety can be replaced with sterically and electronically similarstructures, such as aza-sugars and carbocyclic sugar analogs. Examplesof modifications in a base moiety include alkylated purines andpyrimidines, acylated purines or pyrimidines, or other well-knownheterocyclic substitutes. Nucleic acid monomers can be linked byphosphodiester bonds or analogs of such linkages. Analogs ofphosphodiester linkages include phosphorothioate, phosphorodithioate,phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate,phosphoranilidate, phosphoramidate, and the like. The term “nucleic acidmolecule” also includes so-called “peptide nucleic acids,” whichcomprise naturally-occurring or modified nucleic acid bases attached toa polyamide backbone. Nucleic acids can be either single stranded ordouble stranded.

The term “complement of a nucleic acid molecule” refers to a nucleicacid molecule having a complementary nucleotide sequence and reverseorientation as compared to a reference nucleotide sequence. For example,the sequence 5′ ATGCACGGG 3′ is complementary to 5′ CCCGTGCAT 3′.

The term “degenerate nucleotide sequence” denotes a sequence ofnucleotides that includes one or more degenerate codons as compared to areference nucleic acid molecule that encodes a polypeptide. Degeneratecodons contain different triplets of nucleotides, but encode the sameamino acid residue (i.e., GAU and GAC triplets each encode Asp).

The term “structural gene” refers to a nucleic acid molecule that istranscribed into messenger RNA (mRNA), which is then translated into asequence of amino acids characteristic of a specific polypeptide.

An “isolated nucleic acid molecule” is a nucleic acid molecule that isnot integrated in the genomic DNA of an organism. For example, a DNAmolecule that encodes a growth factor that has been separated from thegenomic DNA of a cell is an isolated DNA molecule. Another example of anisolated nucleic acid molecule is a chemically-synthesized nucleic acidmolecule that is not integrated in the genome of an organism. A nucleicacid molecule that has been isolated from a particular species issmaller than the complete DNA molecule of a chromosome from thatspecies.

A “nucleic acid molecule construct” is a nucleic acid molecule, eithersingle- or double-stranded, that has been modified through humanintervention to contain segments of nucleic acid combined and juxtaposedin an arrangement not existing in nature.

“Linear DNA” denotes non-circular DNA molecules having free 5′ and 3′ends. Linear DNA can be prepared from closed circular DNA molecules,such as plasmids, by enzymatic digestion or physical disruption.

“Complementary DNA (cDNA)” is a single-stranded DNA molecule that isformed from an mRNA template by the enzyme reverse transcriptase.Typically, a primer complementary to portions of mRNA is employed forthe initiation of reverse transcription. Those skilled in the art alsouse the term “cDNA” to refer to a double-stranded DNA moleculeconsisting of such a single-stranded DNA molecule and its complementaryDNA strand. The term “cDNA” also refers to a clone of a cDNA moleculesynthesized from an RNA template.

A “promoter” is a nucleotide sequence that directs the transcription ofa structural gene. Typically, a promoter is located in the 5′ non-codingregion of a gene, proximal to the transcriptional start site of astructural gene. Sequence elements within promoters that function in theinitiation of transcription are often characterized by consensusnucleotide sequences. These promoter elements include RNA polymerasebinding sites, TATA sequences, CAAT sequences, differentiation-specificelements (DSEs; McGehee et al., Mol. Endocrinol. 7:551 (1993)), cyclicAMP response elements (CREs), serum response elements (SREs; Treisman,Seminars in Cancer Biol. 1:47 (1990)), glucocorticoid response elements(GREs), and binding sites for other transcription factors, such asCRE/ATF (O'Reilly et al., J. Biol. Chem. 267:19938 (1992)), AP2 (Ye etal., J. Biol. Chem. 269:25728 (1994)), SP1, cAMP response elementbinding protein (CREB; Loeken, Gene Expr. 3:253 (1993)) and octamerfactors (see, in general, Watson et al., eds., Molecular Biology of theGene, 4th ed. (The Benjamin/Cummings Publishing Company, Inc. 1987), andLemaigre and Rousseau, Biochem. J. 303:1 (1994)). If a promoter is aninducible promoter, then the rate of transcription increases in responseto an inducing agent. In contrast, the rate of transcription is notregulated by an inducing agent if the promoter is a constitutivepromoter. Repressible promoters are also known.

A “core promoter” contains essential nucleotide sequences for promoterfunction, including the TATA box and start of transcription. By thisdefinition, a core promoter may or may not have detectable activity inthe absence of specific sequences that may enhance the activity orconfer tissue specific activity.

A “regulatory element” is a nucleotide sequence that modulates theactivity of a core promoter. For example, a regulatory element maycontain a nucleotide sequence that binds with cellular factors enablingtranscription exclusively or preferentially in particular cells,tissues, or organelles. These types of regulatory elements are normallyassociated with genes that are expressed in a “cell-specific,”“tissue-specific,” or “organelle-specific” manner.

An “enhancer” is a type of regulatory element that can increase theefficiency of transcription, regardless of the distance or orientationof the enhancer relative to the start site of transcription.

“Heterologous DNA” refers to a DNA molecule, or a population of DNAmolecules, that does not exist naturally within a given host cell. DNAmolecules heterologous to a particular host cell may contain DNA derivedfrom the host cell species (i.e., endogenous DNA) so long as that hostDNA is combined with non-host DNA (i.e., exogenous DNA). For example, aDNA molecule containing a non-host DNA segment encoding a polypeptideoperably linked to a host DNA segment comprising a transcriptionpromoter is considered to be a heterologous DNA molecule. Conversely, aheterologous DNA molecule can comprise an endogenous gene operablylinked with an exogenous promoter. As another illustration, a DNAmolecule comprising a gene derived from a wild-type cell is consideredto be heterologous DNA if that DNA molecule is introduced into a mutantcell that lacks the wild-type gene.

A “polypeptide” is a polymer of amino acid residues joined by peptidebonds, whether produced naturally or synthetically. Polypeptides of lessthan about 10 amino acid residues are commonly referred to as“peptides.”

A “protein” is a macromolecule comprising one or more polypeptidechains. A protein may also comprise non-peptidic components, such ascarbohydrate groups. Carbohydrates and other non-peptidic substituentsmay be added to a protein by the cell in which the protein is produced,and will vary with the type of cell. Proteins are defined herein interms of their amino acid backbone structures; substituents such ascarbohydrate groups are generally not specified, but may be presentnonetheless.

A peptide or polypeptide encoded by a non-host DNA molecule is a“heterologous” peptide or polypeptide.

A “cloning vector” is a nucleic acid molecule, such as a plasmid,cosmid, or bacteriophage, that has the capability of replicatingautonomously in a host cell. Cloning vectors typically contain one or asmall number of restriction endonuclease recognition sites that allowinsertion of a nucleic acid molecule in a determinable fashion withoutloss of an essential biological function of the vector, as well asnucleotide sequences encoding a marker gene that is suitable for use inthe identification and selection of cells transformed with the cloningvector. Marker genes typically include genes that provide tetracyclineresistance or ampicillin resistance.

An “expression vector” is a nucleic acid molecule encoding a gene thatis expressed in a host cell. Typically, an expression vector comprises atranscription promoter, a gene, and a transcription terminator. Geneexpression is usually placed under the control of a promoter, and such agene is said to be “operably linked to” the promoter. Similarly, aregulatory element and a core promoter are operably linked if theregulatory element modulates the activity of the core promoter.

A “recombinant host” is a cell that contains a heterologous nucleic acidmolecule, such as a cloning vector or expression vector. In the presentcontext, an example of a recombinant host is a cell that produces anantagonist of the present invention from an expression vector. Incontrast, such an antagonist can be produced by a cell that is a“natural source” of said antagonist, and that lacks an expressionvector.

“Integrative transformants” are recombinant host cells, in whichheterologous DNA has become integrated into the genomic DNA of thecells.

A “fusion protein” is a hybrid protein expressed by a nucleic acidmolecule comprising nucleotide sequences of at least two genes. Forexample, a fusion protein can comprise at least part of a IL-17RApolypeptide fused with a polypeptide that binds an affinity matrix. Sucha fusion protein provides a means to isolate large quantities of IL-17RAusing affinity chromatography.

The term “receptor” denotes a cell-associated protein that binds to abioactive molecule termed a “ligand.” This interaction mediates theeffect of the ligand on the cell. Receptors can be membrane bound,cytosolic or nuclear; monomeric (e.g., thyroid stimulating hormonereceptor, beta-adrenergic receptor) or multimeric (e.g., PDGF receptor,growth hormone receptor, IL-3 receptor, GM-CSF receptor, G-CSF receptor,erythropoietin receptor and IL-6 receptor). Membrane-bound receptors arecharacterized by a multi-domain structure comprising an extracellularligand-binding domain and an intracellular effector domain that istypically involved in signal transduction. In certain membrane-boundreceptors, the extracellular ligand-binding domain and the intracellulareffector domain are located in separate polypeptides that comprise thecomplete functional receptor.

In general, the binding of ligand to receptor results in aconformational change in the receptor that causes an interaction betweenthe effector domain and other molecule(s) in the cell, which in turnleads to an alteration in the metabolism of the cell. Metabolic eventsthat are often linked to receptor-ligand interactions include genetranscription, phosphorylation, dephosphorylation, increases in cyclicAMP production, mobilization of cellular calcium, mobilization ofmembrane lipids, cell adhesion, hydrolysis of inositol lipids andhydrolysis of phospholipids.

The term “secretory signal sequence” denotes a DNA sequence that encodesa peptide (a “secretory peptide”) that, as a component of a largerpolypeptide, directs the larger polypeptide through a secretory pathwayof a cell in which it is synthesized. The larger polypeptide is commonlycleaved to remove the secretory peptide during transit through thesecretory pathway.

An “isolated polypeptide” is a polypeptide that is essentially free fromcontaminating cellular components, such as carbohydrate, lipid, or otherproteinaceous impurities associated with the polypeptide in nature.Typically, a preparation of isolated polypeptide contains thepolypeptide in a highly purified form, i.e., at least about 80% pure, atleast about 90% pure, at least about 95% pure, greater than 95% pure,such as 96%, 97%, or 98% or more pure, or greater than 99% pure. One wayto show that a particular protein preparation contains an isolatedpolypeptide is by the appearance of a single band following sodiumdodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of the proteinpreparation and Coomassie Brilliant Blue staining of the gel. However,the term “isolated” does not exclude the presence of the samepolypeptide in alternative physical forms, such as dimers oralternatively glycosylated or derivatized forms.

The terms “amino-terminal” and “carboxyl-terminal” are used herein todenote positions within polypeptides. Where the context allows, theseterms are used with reference to a particular sequence or portion of apolypeptide to denote proximity or relative position. For example, acertain sequence positioned carboxyl-terminal to a reference sequencewithin a polypeptide is located proximal to the carboxyl terminus of thereference sequence, but is not necessarily at the carboxyl terminus ofthe complete polypeptide.

The term “expression” refers to the biosynthesis of a gene product. Forexample, in the case of a structural gene, expression involvestranscription of the structural gene into mRNA and the translation ofmRNA into one or more polypeptides.

As used herein, the term “immunomodulator” includes cytokines, stem cellgrowth factors, lymphotoxins, co-stimulatory molecules, hematopoieticfactors, and the like, and synthetic analogs of these molecules.

The term “complement/anti-complement pair” denotes non-identicalmoieties that form a non-covalently associated, stable pair underappropriate conditions. For instance, biotin and avidin (orstreptavidin) are prototypical members of a complement/anti-complementpair. Other exemplary complement/anti-complement pairs includereceptor/ligand pairs, antibody/antigen (or hapten or epitope) pairs,sense/antisense polynucleotide pairs, and the like. Where subsequentdissociation of the complement/anti-complement pair is desirable, thecomplement/anti-complement pair preferably has a binding affinity ofless than 10⁹ M⁻¹.

As used herein, a “therapeutic agent” is a molecule or atom which isconjugated to an antibody moiety to produce a conjugate which is usefulfor therapy. Examples of therapeutic agents include drugs, toxins,immunomodulators, chelators, boron compounds, photoactive agents ordyes, and radioisotopes.

A “detectable label” is a molecule or atom which can be conjugated to anantibody moiety to produce a molecule useful for diagnosis. Examples ofdetectable labels include chelators, photoactive agents, radioisotopes,fluorescent agents, paramagnetic ions, or other marker moieties.

The term “affinity tag” is used herein to denote a polypeptide segmentthat can be attached to a second polypeptide to provide for purificationor detection of the second polypeptide or provide sites for attachmentof the second polypeptide to a substrate. In principal, any peptide orprotein for which an antibody or other specific binding agent isavailable can be used as an affinity tag. Affinity tags include apoly-histidine tract, protein A (Nilsson et al., EMBO J. 4:1075 (1985);Nilsson et al., Methods Enzymol. 198:3 (1991)), glutathione Stransferase (Smith and Johnson, Gene 67:31 (1988)), Glu-Glu affinity tag(Grussenmeyer et al., Proc. Natl. Acad. Sci. USA 82:7952 (1985)),substance P, FLAG peptide (Hopp et al., Biotechnology 6:1204 (1988)),streptavidin binding peptide, or other antigenic epitope or bindingdomain. See, in general, Ford et al., Protein Expression andPurification 2:95 (1991). DNA molecules encoding affinity tags areavailable from commercial suppliers (e.g., Pharmacia Biotech,Piscataway, N.J.).

A “target polypeptide” or a “target peptide” is an amino acid sequencethat comprises at least one epitope, and that is expressed on a targetcell, such as a tumor cell, or a cell that carries an infectious agentantigen. T cells recognize peptide epitopes presented by a majorhistocompatibility complex molecule to a target polypeptide or targetpeptide and typically lyse the target cell or recruit other immune cellsto the site of the target cell, thereby killing the target cell.

In eukaryotes, RNA polymerase II catalyzes the transcription of astructural gene to produce mRNA. A nucleic acid molecule can be designedto contain an RNA polymerase II template in which the RNA transcript hasa sequence that is complementary to that of a specific mRNA. The RNAtranscript is termed an “anti-sense RNA” and a nucleic acid moleculethat encodes the anti-sense RNA is termed an “anti-sense gene.”Anti-sense RNA molecules are capable of binding to mRNA molecules,resulting in an inhibition of mRNA translation.

An “anti-sense oligonucleotide specific for IL-17A or IL-23” is anoligonucleotide having a sequence (a) capable of forming a stabletriplex with a portion of the IL-17A or IL-23 gene, or (b) capable offorming a stable duplex with a portion of an mRNA transcript of theIL-17A or IL-23 gene.

A “ribozyme” is a nucleic acid molecule that contains a catalyticcenter. The term includes RNA enzymes, self-splicing RNAs, self-cleavingRNAs, and nucleic acid molecules that perform these catalytic functions.A nucleic acid molecule that encodes a ribozyme is termed a “ribozymegene.”

An “external guide sequence” is a nucleic acid molecule that directs theendogenous ribozyme, RNase P, to a particular species of intracellularmRNA, resulting in the cleavage of the mRNA by RNase P. A nucleic acidmolecule that encodes an external guide sequence is termed an “externalguide sequence gene.”

The term “allelic variant” is used herein to denote any of two or morealternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inphenotypic polymorphism within populations. Gene mutations can be silent(no change in the encoded polypeptide) or may encode polypeptides havingaltered amino acid sequence. The term allelic variant is also usedherein to denote a protein encoded by an allelic variant of a gene.

The term “ortholog” denotes a polypeptide or protein obtained from onespecies that is the functional counterpart of a polypeptide or proteinfrom a different species. Sequence differences among orthologs are theresult of speciation.

“Paralogs” are distinct but structurally related proteins made by anorganism. Paralogs are believed to arise through gene duplication. Forexample, α-globin, β-globin, and myoglobin are paralogs of each other.

Due to the imprecision of standard analytical methods, molecular weightsand lengths of polymers are understood to be approximate values. Whensuch a value is expressed as “about” X or “approximately” X, the statedvalue of X will be understood to be accurate to ±10%.

C) Antibodies that Bind IL-17 and IL-23 (Via p19)

The antibodies of the invention specifically bind to IL-17 and IL-23(via p19). In some embodiments, the antibodies of the inventionspecifically bind a monomeric form of both IL-17 and IL-23. In someembodiments, the antibodies of the invention bind a homodimeric form ofeither IL-17 or IL-23. In still other embodiments, the antibodies of theinvention specifically bind a multimeric form of IL-17 or IL-23 (e.g., aheterodimeric form). For instance, IL-17 can form a heterodimer with anyother member of the IL-17 family of ligands, such as IL-17B, IL-17C, orIL-17F. Preferred antibodies of the invention block the biologicalactivities of IL-17 and IL-23, either singly or together.

Preferred antibodies, and antibodies suitable for use in the method ofthe invention, include, for example, fully human antibodies, humanantibody homologs, humanized antibody homologs, chimeric antibodyhomologs, Fab, Fab′, F(ab).sub.2, F(ab′).sub.2 and F(v) antibodyfragments, single chain antibodies, and monomers or dimers of antibodyheavy or light chains or mixtures thereof. Antibodies of the inventionare preferably monoclonal antibodies.

The antibodies of the invention may include intact immunoglobulins ofany isotype including types IgA, IgG, IgE, IgD, IgM (as well as subtypesthereof). The antibodies preferably include intact IgG and morepreferably IgG1. The light chains of the immunoglobulin may be kappa orlambda. The light chains are preferably kappa.

The antibodies of the invention comprise or consist of portions ofintact antibodies that retain antigen-binding specificity, for example,Fab fragments, Fab′ fragments, F(ab′).sub.2 fragments, F(v) fragments,heavy chain monomers or dimers, light chain monomers or dimers, dimersconsisting of one heavy and one light chain, and the like. Thus, antigenbinding fragments, as well as full-length dimeric or trimericpolypeptides derived from the above-described antibodies are themselvesuseful.

The direct use of rodent monoclonal antibodies (MAbs) as humantherapeutic agents led to human anti-rodent antibody (“HARA”) (forexample, human anti-mouse antibody (“HAMA”)) responses which occurred ina significant number of patients treated with the rodent-derivedantibody (Khazaeli, et al., (1994) Immunother. 15:42-52). Chimericantibodies containing fewer murine amino acid sequences are believed tocircumvent the problem of eliciting an immune response in humans.

Refinement of antibodies to avoid the problem of HARA responses led tothe development of “humanized antibodies.” Humanized antibodies areproduced by recombinant DNA technology, in which at least one of theamino acids of a human immunoglobulin light or heavy chain that is notrequired for antigen binding has been substituted for the correspondingamino acid from a nonhuman mammalian immunoglobulin light or heavychain. For example, if the immunoglobulin is a mouse monoclonalantibody, at least one amino acid that is not required for antigenbinding is substituted using the amino acid that is present on acorresponding human antibody in that position. Without wishing to bebound by any particular theory of operation, it is believed that the“humanization” of the monoclonal antibody inhibits human immunologicalreactivity against the foreign immunoglobulin molecule.

As a non-limiting example, a method of performing complementaritydetermining region (CDR) grafting may be performed by sequencing themouse heavy and light chains of the antibody of interest that binds tothe target antigen (e.g., IL-17 and/or IL-23/p19) and geneticallyengineering the CDR DNA sequences and imposing these amino acidsequences to corresponding human V regions by site directed mutagenesis.Human constant region gene segments of the desired isotype are added,and the “humanized” heavy and light chain genes are co-expressed inmammalian cells to produce soluble humanized antibody. A typicalexpression cell is a Chinese Hamster Ovary (CHO) cell. Suitable methodsfor creating the chimeric antibodies may be found, for example, in Joneset al. (1986) Nature 321:522-525; Riechmann (1988) Nature 332:323-327;Queen et al. (1989) Proc. Nat. Acad. Sci. USA 86:10029; and Orlandi etal (1989) Proc. Natl. Acad. Sci. USA 86:3833.

Queen et al. (1989) Proc. Nat. Acad. Sci. USA 86:10029-10033 and WO90/07861 describe the preparation of a humanized antibody. Human andmouse variable framework regions were chosen for optimal proteinsequence homology. The tertiary structure of the murine variable regionwas computer-modeled and superimposed on the homologous human frameworkto show optimal interaction of amino acid residues with the mouse CDRs.This led to the development of antibodies with improved binding affinityfor antigen (which is typically decreased upon making CDR-graftedchimeric antibodies). Alternative approaches to making humanizedantibodies are known in the art and are described, for example, inTempest (1991) Biotechnology 9:266-271.

The antibodies of the invention may be used alone or as immunoconjugateswith a cytotoxic agent. In some embodiments, the agent is achemotherapeutic agent. In some embodiments, the agent is aradioisotope, including, but not limited to Lead-212, Bismuth-212,Astatine-211, Iodine-131, Scandium-47, Rhenium-186, Rhenium-188,Yttrium-90, Iodine-123, Iodine-125, Bromine-77, Indium-111, andfissionable nuclides such as Boron-10 or an Actinide. In otherembodiments, the agent is a toxin or cytotoxic drug, including but notlimited to ricin, modified Pseudomonas enterotoxin A, calicheamicin,adriamycin, 5-fluorouracil, and the like. Methods of conjugation ofantibodies and antibody fragments to such agents are known in theliterature.

The antibodies of the invention include derivatives that are modified,e.g., by the covalent attachment of any type of molecule to the antibodysuch that covalent attachment does not prevent the antibody from bindingto its epitope. Examples of suitable derivatives include, but are notlimited to fucosylated antibodies and fragments, glycosylated antibodiesand fragments, acetylated antibodies and fragments, pegylated antibodiesand fragments, phosphorylated antibodies and fragments, and amidatedantibodies and fragments. The antibodies and derivatives thereof of theinvention may themselves by derivatized by known protecting/blockinggroups, proteolytic cleavage, linkage to a cellular ligand or otherproteins, and the like. In some embodiments of the invention, at leastone heavy chain of the antibody is fucosylated. In some embodiments, thefucosylation is N-linked. In some preferred embodiments, at least oneheavy chain of the antibody comprises a fucosylated, N-linkedoligosaccharide.

The antibodies of the invention include variants having single ormultiple amino acid substitutions, deletions, additions, or replacementsthat retain, the biological properties (e.g., block the binding of IL-17and/or IL-23 to their respective receptors, block the biologicalactivity of IL-17 and IL-23, binding affinity) of the antibodies of theinvention. The skilled person can produce variants having single ormultiple amino acid substitutions, deletions, additions or replacements.These variants may include, inter alia: (a) variants in which one ormore amino acid residues are substituted with conservative ornonconservative amino acids, (b) variants in which one or more aminoacids are added to or deleted from the polypeptide, (c) variants inwhich one or more amino acids include a substituent group, and (d)variants in which the polypeptide is fused with another peptide orpolypeptide such as a fusion partner, a protein tag or other chemicalmoiety, that may confer useful properties to the polypeptide, such as,for example, an epitope for an antibody, a polyhistidine sequence, abiotin moiety and the like. Antibodies of the invention may includevariants in which amino acid residues from one species are substitutedfor the corresponding residue in another species, either at theconserved or non conserved positions. In another embodiment, amino acidresidues at nonconserved positions are substituted with conservative ornonconservative residues. The techniques for obtaining these variants,including genetic (suppressions, deletions, mutations, etc.), chemical,and enzymatic techniques, are known to the person having ordinary skillin the art. Antibodies of the invention also include antibody fragments.A “fragment” refers to polypeptide sequences which are at least about40, at least to about 50, at least about 60, at least about 70, at leastabout 80, at least about 90, and at least about 100 amino acids inlength, and which retain some biological activity or immunologicalactivity of the full-length sequence, such as, for example, the abilityto block the binding of IL-17 and/or IL-23 to their respectivereceptors, block the biological activity of IL-17 and IL-23, and affectbinding affinity.

The invention also encompasses fully human antibodies such as thosederived from peripheral blood mononuclear cells of ovarian, breast,renal, colorectal, lung, endometrial, or brain cancer patients. Suchcells may be fused with myeloma cells, for example, to form hybridomacells producing fully human antibodies against both IL-17 and IL-23/p19.

The invention also encompasses bispecific antibodies that bind to bothIL-17 and IL-23 (via p19).

Antibodies that bind to IL-17A and IL-23p19 have been identified byscreening a phage display library. Methods of screening by phage displayare described in detail in standard reference texts, such as Babas,Phage Display: A Laboratory Manual Cold Spring Harbor Lab Press, 2001and Lo, Benny K. C., A., Antibody Engineering, 2004. Such phage displaylibraries can be used to display expressed proteins on the surface of acell or other substance such that the complementary binding entity canbe functionally isolated. In one such phage display library, theantibody light-chain variable region and a portion of the heavy-chainvariable region are combined with synthetic DNA encoding human antibodysequences, which are then displayed on phage and phagemid libraries asFab antibody fragments (Dyax® Human Antibody Libraries, Dyax Corp.,Cambridge, Mass.). Thus, the variable light and heavy chain fragments ofantibodies can be isolated in a Fab format. These variable regions canthen be manipulated to generate antibodies, including antigen-bindingfragments, such as scFvs, bispecific scFvs and multispecific,multifunctional antagonists to IL-17A or IL-23p19.

Using this technology the variable regions of Fabs have been identifiedfor their characteristics of binding and or neutralizing either IL-17Aor IL-23p19 in the plate-based assays described in Examples 15 through18 herein. These variable regions were manipulated to generate variousbinding entities, including scFvs that bind and/or neutralize IL-17A orIL-23p19.

Table 1 below shows a list of the Fabs or scFvs that bind IL-17A.

TABLE 1 Light Light Light Light Light Light Light Heavy Heavy HeavyHeavy Heavy Heavy Heavy VL nucleotide VH nucleotide VL polypeptide VHpolypeptide FR1 CDR1 FR2 CDR2 FR3 CDR3 FR4 FR1 CDR1 FR2 CDR2 FR3 CDR3FR4 Cluster # SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: range rangerange range range range range range range range range range range range83 78 79 80 81 1-22 23-33 34-48 49-55 56-87 88-96 97-106 1-30 31-3536-49 50-66 67-98 99-116 117-127 84 82 83 84 85 1-22 23-35 36-50 51-5758-89  90-100 101-110  1-30 31-35 36-49 50-66 67-98 99-118 119-129 85 8687 88 89 1-23 24-34 35-49 50-56 57-88 89-97 98-107 1-30 31-35 36-4950-67 68-99 100-115  116-126 86 90 91 92 93 1-23 24-34 35-49 50-56 57-8889-97 98-107 1-30 31-35 36-49 50-66 67-98 99-113 114-124 87 94 95 96 971-23 24-34 35-49 50-56 57-88 89-97 98-107 1-30 31-35 36-49 50-66 67-9899-108 109-119 88 98 99 100 101 1-23 24-34 35-49 50-56 57-88 89-9798-107 1-30 31-35 36-49 50-66 67-98 99-114 115-125 89 102 103 104 1051-22 23-33 34-48 49-55 56-87 88-96 97-106 1-30 31-35 36-49 50-66 67-9899-112 113-123 90 106 107 108 109 1-23 24-34 35-49 50-56 57-88 89-9798-107 1-30 31-35 36-49 50-66 67-98 99-114 115-125 91 110 111 112 1131-23 24-34 35-49 50-56 57-88 89-97 98-107 1-30 31-35 36-49 50-66 67-9899-103 104-114 92 114 115 116 117 1-23 24-34 35-49 50-56 57-88 89-9798-107 1-30 31-35 36-49 50-66 67-98 99-107 108-118 93 118 119 120 1211-23 24-39 40-54 55-61 62-93  94-103 104-113  1-30 31-35 36-49 50-6667-98 99-105 106-116 94 122 123 124 125 1-23 24-34 35-49 50-56 57-8889-97 98-107 1-30 31-35 36-49 50-66 67-98 99-114 115-125 95 126 127 128129 1-23 24-34 35-49 50-56 57-88 89-97 98-107 1-30 31-35 36-49 50-6667-98 99-114 115-125 96 130 131 132 133 1-23 24-34 35-49 50-56 57-8889-97 98-107 1-30 31-35 36-49 50-66 67-98 99-105 106-116 97 134 135 136137 1-23 24-34 35-49 50-56 57-88 89-97 98-107 1-30 31-35 36-49 50-6667-98 99-113 114-124 98 138 139 140 141 1-23 24-34 35-49 50-56 57-8889-97 98-107 1-30 31-35 36-49 50-66 67-98 99-106 107-117 99 142 143 144145 1-22 23-33 34-48 49-55 56-87 88-97 98-107 1-30 31-35 36-49 50-6667-98 99-111 112-122 100 146 147 148 149 1-23 24-34 35-49 50-56 57-8889-97 98-107 1-30 31-35 36-49 50-66 67-98 99-112 113-123 191 150 151 152153 1-23 24-34 35-49 50-56 57-88 89-97 98-107 1-30 31-35 36-49 50-6667-98 99-113 114-124 192 154 155 156 157 1-22 23-33 34-48 49-55 56-8788-97 98-107 1-30 31-35 36-49 50-66 67-98 99-111 112-122 193 158 159 160161 1-22 23-33 34-48 49-55 56-87 88-97 98-107 1-30 31-35 36-49 50-6667-98 99-111 112-122 195 162 163 164 165 1-22 23-33 34-48 49-55 56-8788-97 98-107 1-30 31-35 36-49 50-66 67-98 99-111 112-122 196 166 167 168169 1-22 23-33 34-48 49-55 56-87 88-97 98-107 1-30 31-35 36-49 50-6667-98 99-111 112-122 199 170 171 172 173 1-23 24-34 35-49 50-56 57-8889-97 98-107 1-30 31-35 36-49 50-66 67-98 99-106 107-117 200 174 175 176177 1-23 24-34 35-49 50-56 57-88 89-97 98-107 1-30 31-35 36-49 50-6667-98 99-108 109-119 202 178 179 180 181 1-23 24-34 35-49 50-56 57-8889-97 98-107 1-30 31-35 36-49 50-66 67-98 99-113 114-124 203 182 183 184185 1-22 23-33 34-48 49-55 56-87 88-97 98-107 1-30 31-35 36-49 50-6667-98 99-111 112-122 204 186 187 188 189 1-23 24-34 35-49 50-56 57-8889-97 98-107 1-30 31-35 36-49 50-66 67-98 99-108 109-119 207 190 191 192193 1-23 24-34 35-49 50-56 57-88 89-97 98-107 1-30 31-35 36-49 50-6667-98 99-113 114-124 209 194 195 196 197 1-23 24-34 35-49 50-56 57-8889-97 98-107 1-30 31-35 36-49 50-66 67-98 99-108 109-119 210 198 199 200201 1-22 23-33 34-48 49-55 56-87 88-97 98-107 1-30 31-35 36-49 50-6667-98 99-111 112-122 212 202 203 204 205 1-23 24-34 35-49 50-56 57-8889-97 98-107 1-30 31-35 36-49 50-66 67-98 99-113 114-124 213 206 207 208209 1-23 24-34 35-49 50-56 57-88 89-97 98-107 1-30 31-35 36-49 50-6667-98 99-112 113-123 214 210 211 212 213 1-23 24-34 35-49 50-56 57-8889-97 98-107 1-30 31-35 36-49 50-66 67-98 99-113 114-124 218 214 215 216217 1-22 23-33 34-48 49-55 56-87 88-97 98-107 1-30 31-35 36-49 50-6667-98 99-111 112-122 219 218 219 220 221 1-22 23-33 34-48 49-55 56-8788-97 98-107 1-30 31-35 36-49 50-66 67-98 99-112 113-123 220 222 223 224225 1-22 23-33 34-48 49-55 56-87 88-97 98-107 1-30 31-35 36-49 50-6667-98 99-111 112-122 221 226 227 228 229 1-22 23-33 34-48 49-55 56-8788-97 98-107 1-30 31-35 36-49 50-66 67-98 99-106 107-117 222 230 231 232233 1-23 24-34 35-49 50-56 57-88 89-97 98-107 1-30 31-35 36-49 50-6667-98 99-106 107-117 223 234 235 236 237 1-23 24-34 35-49 50-56 57-8889-97 98-107 1-30 31-35 36-49 50-66 67-98 99-108 109-119 224 238 239 240241 1-23 24-34 35-49 50-56 57-88 89-97 98-107 1-30 31-35 36-49 50-6667-98 99-106 107-117 225 242 243 244 245 1-22 23-33 34-48 49-55 56-8788-97 98-107 1-30 31-35 36-49 50-66 67-98 99-111 112-122 226 246 247 248249 1-23 24-34 35-49 50-56 57-88 89-97 98-107 1-30 31-35 36-49 50-6667-98 99-108 109-119 227 250 251 252 253 1-22 23-33 34-48 49-55 56-8788-97 98-107 1-30 31-35 36-49 50-66 67-98 99-111 112-122 229 254 255 256257 1-23 24-34 35-49 50-56 57-88 89-97 98-107 1-30 31-35 36-49 50-6667-98 99-114 115-125 230 258 259 260 261 1-23 24-34 35-49 50-56 57-8889-97 98-107 1-30 31-35 36-49 50-66 67-98 99-106 107-117 231 262 263 264265 1-22 23-33 34-48 49-55 56-87 88-97 98-107 1-27 28-32 33-46 47-6364-95 96-103 104-114 232 266 267 268 269 1-23 24-34 35-49 50-56 57-8889-97 98-107 1-30 31-35 36-49 50-66 67-98 99-106 107-117 233 270 271 272273 1-23 24-34 35-49 50-56 57-88 89-97 98-107 1-30 31-35 36-49 50-6667-98 99-106 107-117 234 274 275 276 277 1-23 24-34 35-49 50-56 57-8889-97 98-107 1-30 31-35 36-49 50-66 67-98 99-108 109-119 235 278 279 280281 1-22 23-33 34-48 49-55 56-87 88-97 98-107 1-30 31-35 36-49 50-6667-98 99-111 112-122 236 282 283 284 285 1-23 24-34 35-49 50-56 57-8889-97 98-107 1-30 31-35 36-49 50-66 67-98 99-111 112-122 237 286 287 288289 1-22 23-33 34-48 49-55 56-87 88-97 98-107 1-30 31-35 36-49 50-6667-98 99-111 112-122 238 290 291 292 293 1-21 22-32 33-47 48-54 55-8687-96 97-106 1-30 31-35 36-49 50-66 67-98 99-111 112-122 239 294 295 296297 1-22 23-33 34-48 49-55 56-87 88-97 98-107 1-30 31-35 36-49 50-6667-98 99-111 112-122 240 298 299 300 301 1-22 23-33 34-48 49-55 56-8788-97 98-107 1-30 31-35 36-49 50-66 67-98 99-111 112-122 245 302 303 304305 1-22 23-33 34-48 49-55 56-87 88-97 98-107 1-30 31-35 36-49 50-6667-98 99-111 112-122 246 306 307 308 309 1-23 24-34 35-49 50-56 57-8889-97 98-107 1-30 31-35 36-49 50-66 67-98 99-106 107-117 248 310 311 312313 1-22 23-33 34-48 49-55 56-87 88-97 98-107 1-30 31-35 36-49 50-6667-98 99-111 112-122 249 314 315 316 317 1-22 23-33 34-48 49-55 56-8788-97 98-107 1-28 29-33 34-47 48-64 65-96 97-112 113-123 250 318 319 320321 1-23 24-34 35-49 50-56 57-88 89-97 98-107 1-30 31-35 36-49 50-6667-98 99-112 113-123 296 None 322 None 323 — — — — — — — 1-30 31-3536-49 50-66 67-98 99-116 117-127 297 324 None 325 None 1-22 23-35 36-5051-57 58-89  90-100 101-110  — — — — — — —

Table 2 below shows a list of the Fabs or scFvs that bind IL-23p19

TABLE 2 Light Light Light Light Light Light Light Heavy Heavy HeavyHeavy Heavy Heavy Heavy VL nucleotide VH nucleotide VL polypeptide VHpolypeptide FR1 CDR1 FR2 CDR2 FR3 CDR3 FR4 FR1 CDR1 FR2 CDR2 FR3 CDR3FR4 Cluster # SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: range rangerange range range range range range range range range range range range26 492 493 754 755 1-23 24-35 36-50 51-57 58-89 90-98   99-108 1-3031-35 36-49 50-66 67-98 99-105 106-116 27 494 495 756 757 1-23 24-3435-49 50-56 57-88 89-97   98-107 1-30 31-35 36-49 50-66 67-98 99-106107-117 28 496 497 758 759 1-23 24-35 36-50 51-57 58-89 90-98   99-1081-30 31-35 36-49 50-66 67-98 99-108 109-119 29 498 499 460 761 1-2223-36 37-51 52-58 59-90 91-102 103-112 1-30 31-35 36-49 50-66 67-9899-112 113-123 33 500 501 762 763 1-23 24-34 35-49 50-56 57-88 89-97  98-107 1-30 31-35 36-49 50-66 67-98 99-113 114-124 36 502 503 764 7651-23 24-34 35-49 50-56 57-88 89-97   98-107 1-30 31-35 36-49 50-66 67-9899-105 106-116 40 504 505 766 767 1-22 23-33 34-48 49-55 56-87 88-96  97-106 1-30 31-35 36-49 50-66 67-98 99-116 117-127 41 506 507 768 7691-23 24-39 40-54 55-61 62-93 94-102 103-112 1-30 31-35 36-49 50-66 67-9899-112 113-123 43 508 509 770 771 1-23 24-39 40-54 55-61 62-93 94-102103-112 1-30 31-35 36-49 50-66 67-98 99-107 108-118 101 510 511 772 7731-22 23-36 37-51 52-58 59-90 91-102 103-112 1-30 31-35 36-49 50-66 67-9899-112 113-123 102 512 513 774 775 1-23 24-34 35-49 50-56 57-88 89-97  98-107 1-30 31-35 36-49 50-66 67-98 99-110 111-121 103 514 515 776 7771-23 24-39 40-54 55-61 62-93 94-102 103-112 1-30 31-35 36-49 50-66 67-9899-113 114-124 110 516 517 778 779 1-22 23-35 36-50 51-57 58-89 90-98  99-108 1-30 31-35 36-49 50-66 67-98 99-119 120-130 114 518 519 780 7811-23 24-34 35-49 50-56 57-88 89-97   98-107 1-30 31-35 36-49 50-66 67-9899-108 109-119 115 520 521 782 783 1-23 24-39 40-54 55-61 62-93 94-102103-112 1-30 31-35 36-49 50-66 67-98 99-108 109-119 119 522 523 784 7851-23 24-34 35-49 50-56 57-88 89-97   98-107 1-30 31-35 36-49 50-66 67-9899-106 107-117 120 524 525 786 78 1-23 24-39 40-54 55-61 62-93 94-102103-112 1-30 31-35 36-49 50-66 67-98 99-108 109-119 121 526 527 788 7891-23 24-34 35-49 50-56 57-88 89-97   98-107 1-30 31-35 36-49 50-66 67-9899-109 110-120 122 528 529 790 791 1-23 24-34 35-49 50-56 57-88 89-96  97-106 1-30 31-35 36-49 50-66 67-98 99-108 109-119 123 530 531 792 7931-22 23-33 34-48 49-55 56-87 88-97   98-107 1-30 31-35 36-49 50-66 67-9899-111 112-122 124 532 533 794 795 1-23 24-34 35-49 50-56 57-88 89-97  98-107 1-30 31-35 36-49 50-66 67-98 99-110 111-121 125 534 535 796 7971-23 24-34 35-49 50-56 57-88 89-97   98-107 1-30 31-35 36-49 50-66 67-9899-106 107-117 126 536 537 798 799 1-22 23-35 36-50 51-57 58-89 90-100101-110 1-30 31-35 36-49 50-66 67-98 99-106 107-117 127 538 539 800 8011-22 23-35 36-50 51-57 58-89 90-100 101-110 1-30 31-35 36-49 50-66 67-9899-111 112-122 128 540 541 802 803 1-22 23-35 36-50 51-57 58-89 90-100101-110 1-30 31-35 36-49 50-66 67-98 99-111 112-122 129 542 543 804 8051-23 24-34 35-49 50-56 57-88 89-98   99-108 1-30 31-35 36-49 50-66 67-9899-107 108-118 130 544 545 806 807 1-23 24-34 35-49 50-56 57-88 89-97  98-107 1-30 31-35 36-49 50-66 67-98 99-109 110-120 131 546 547 808 8091-23 24-34 35-49 50-56 57-88 89-97   98-107 1-30 31-35 36-49 50-66 67-9899-109 110-120 132 548 548 810 811 1-23 24-34 35-49 50-56 57-88 89-97  98-107 1-30 31-35 36-49 50-66 67-98 99-115 116-126 134 550 551 812 8131-22 23-33 34-48 49-55 56-87 88-96   97-106 1-30 31-35 36-49 50-66 67-9899-111 112-122 135 552 553 814 815 1-23 24-34 35-49 50-56 57-88 89-98  99-108 1-30 31-35 36-49 50-66 67-98 99-116 117-127 136 554 555 816 8171-23 24-34 35-49 50-56 57-88 89-98   99-108 1-30 31-35 36-49 50-66 67-9899-109 110-120 137 556 557 818 819 1-22 23-36 37-51 52-58 59-90 91-100101-110 1-30 31-35 36-49 50-66 67-98 99-105 106-116 138 558 559 820 8201-23 24-39 40-54 55-61 62-93 94-102 103-112 1-30 31-35 36-49 50-66 67-9899-107 108-118 139 560 561 822 823 1-23 24-34 35-49 50-56 57-88 89-97  98-107 1-30 31-35 36-49 50-66 67-98 99-111 112-122 140 562 563 824 8251-23 24-39 40-54 55-61 62-93 94-102 103-112 1-30 31-35 36-49 50-66 67-9899-111 112-122 141 564 565 826 827 1-23 24-39 40-54 55-61 62-93 94-102103-112 1-30 31-35 36-49 50-66 67-98 99-107 108-118 142 566 567 828 8291-23 24-34 35-49 50-56 57-88 89-97   98-107 1-30 31-35 36-49 50-66 67-9899-113 114-124 143 568 569 830 831 1-23 24-34 35-49 50-56 57-88 89-97  98-107 1-30 31-35 36-49 50-66 67-98 99-107 108-118 144 570 571 832 8331-23 24-39 40-54 55-61 62-93 94-102 103-112 1-30 31-35 36-49 50-66 67-9899-111 112-122 145 572 573 834 835 1-23 24-34 35-49 50-56 57-88 89-96  97-106 1-30 31-35 36-49 50-66 67-98 99-107 108-118 146 574 575 836 8371-22 23-36 37-51 52-58 59-90 91-100 101-110 1-30 31-35 36-49 50-66 67-9899-111 112-122 148 576 577 838 839 1-23 24-34 35-49 50-56 57-88 89-97  98-107 1-30 31-35 36-49 50-66 67-98 99-105 106-116 149 578 579 840 8411-23 24-34 35-49 50-56 57-88 89-98   99-108 1-30 31-35 36-49 50-66 67-9899-105 106-116 150 580 581 842 843 1-23 24-39 40-54 55-61 62-93 94-101102-111 1-30 31-35 36-49 50-66 67-98 99-108 109-119 151 582 583 844 8451-23 24-34 35-49 50-56 57-88 89-97   98-107 1-30 31-35 36-49 50-66 67-9899-108 109-119 152 584 586 846 847 1-23 24-34 35-49 50-56 57-88 89-98  99-108 1-30 31-35 36-49 50-65 66-97 98-110 111-121 153 586 587 848 8491-23 24-34 35-49 50-56 57-88 89-97   98-107 1-30 31-35 36-49 50-66 67-9899-110 111-121 154 588 589 850 851 1-23 24-39 40-54 55-61 62-93 94-102103-112 1-30 31-35 36-49 50-66 67-98 99-107 108-118 155 590 591 825 8531-23 24-35 36-50 51-57 58-89 90-100 101-110 1-30 31-35 36-49 50-66 67-9899-109 110-120 156 592 593 854 855 1-22 23-35 36-50 51-57 58-89 90-100101-110 1-30 31-35 36-49 50-66 67-98 99-109 110-120 157 594 595 856 8571-22 23-35 36-50 51-57 58-89 90-100 101-110 1-30 31-35 36-49 50-66 67-9899-113 114-124 158 596 597 858 589 1-23 24-39 40-54 55-61 62-93 94-102103-112 1-30 31-35 36-49 50-66 67-98 99-113 114-124 159 598 599 860 8611-23 24-39 40-54 55-61 62-93 94-102 103-112 1-30 31-35 36-49 50-66 67-9899-112 113-123 160 600 601 862 863 1-23 24-34 35-49 50-56 57-88 89-96  97-106 1-30 31-35 36-49 50-66 67-98 99-105 106-116 161 602 603 864 8651-23 24-39 40-54 55-61 62-93 94-102 103-112 1-30 31-35 36-49 50-66 67-9899-107 108-118 162 604 605 866 867 1-23 24-35 36-50 51-57 58-89 90-98  99-108 1-30 31-35 36-49 50-66 67-98 99-105 106-116 163 606 607 868 8691-23 24-35 36-50 51-57 58-89 90-98   99-108 1-30 31-35 36-49 50-66 67-9899-108 109-119 164 608 609 870 871 1-23 24-35 36-50 51-57 58-89 90-98  99-108 1-30 31-35 36-49 50-66 67-98 99-107 108-118 165 610 611 872 8731-23 24-39 40-54 55-61 62-93 94-102 103-112 1-30 31-35 36-49 50-66 67-9899-109 110-120 166 612 613 874 875 1-23 24-35 36-50 51-57 58-89 90-98  99-108 1-30 31-35 36-49 50-66 67-98 99-105 106-116 167 614 615 876 8771-23 24-34 35-49 50-56 57-88 89-97   98-107 1-30 31-35 36-49 50-66 67-9899-103 104-114 168 616 617 878 879 1-23 24-34 35-49 50-56 57-88 89-97  98-107 1-30 31-35 36-49 50-66 67-98 99-108 109-119 169 618 619 880 8811-23 24-35 36-50 51-57 58-89 90-99  100-109 1-30 31-35 36-49 50-66 67-9899-112 113-123 170 620 621 882 883 1-23 24-39 40-54 55-61 62-93 94-101102-111 1-30 31-35 36-49 50-66 67-98 99-112 113-123 171 622 623 884 8851-23 24-35 36-50 51-57 58-89 90-101 102-111 1-30 31-35 36-49 50-66 67-9899-112 113-123 172 624 625 886 887 1-23 24-34 35-49 50-56 57-88 89-96  97-106 1-30 31-35 36-49 50-66 67-98 99-107 108-118 173 626 627 888 8891-23 24-34 35-49 50-56 57-88 89-97   98-107 1-30 31-35 36-49 50-66 67-9899-109 110-120 174 628 629 890 891 1-23 24-34 35-49 50-56 57-88 89-97  98-107 1-30 31-35 36-49 50-66 67-98 99-106 107-117 175 630 631 892 8931-23 24-34 35-49 50-56 57-88 89-99  100-109 1-30 31-35 36-49 50-66 67-9899-112 113-123 176 632 633 894 895 1-23 24-34 35-49 50-56 57-88 89-98  99-108 1-30 31-35 36-49 50-66 67-98 99-109 110-120 178 634 635 896 8971-23 24-39 40-54 55-61 62-93 94-101 102-111 1-30 31-35 36-49 50-66 67-9899-113 114-124 179 636 637 898 899 1-23 24-39 40-54 55-61 62-93 94-102103-112 1-30 31-35 36-49 50-66 67-98 99-110 111-121 180 638 639 900 9011-23 24-34 35-49 50-56 57-88 89-98   99-108 1-30 31-35 36-49 50-66 67-9899-109 110-120 181 640 641 902 903 1-23 24-34 35-49 50-56 57-88 89-97  98-107 1-30 31-35 36-49 50-66 67-98 99-114 115-125 182 642 643 904 9051-23 24-39 40-54 55-61 62-93 94-102 103-112 1-30 31-35 36-49 50-66 67-9899-108 109-119 183 644 645 906 907 1-23 24-35 36-50 51-57 58-89 90-98  99-108 1-30 31-35 36-49 50-66 67-98 99-107 108-118 184 646 647 908 9091-23 24-34 35-49 50-56 57-88 89-97   98-107 1-30 31-35 36-49 50-66 67-9899-117 118-128 185 648 649 910 911 1-23 24-39 40-54 55-61 62-93 94-101102-111 1-30 31-35 36-49 50-66 67-98 99-110 111-121 186 650 651 912 9131-23 24-35 36-50 51-57 58-89 90-99  100-109 1-30 31-35 36-49 50-66 67-9899-107 108-118 187 652 653 914 915 1-23 24-34 35-49 50-56 57-88 89-97  98-107 1-30 31-35 36-49 50-66 67-98 99-107 108-118 188 654 655 916 9171-23 24-39 40-54 55-61 62-93 94-103 104-113 1-30 31-35 36-49 50-66 67-9899-111 112-122 189 565 567 918 919 1-23 24-39 40-54 55-61 62-93 94-102103-112 1-30 31-35 36-49 50-66 67-98 99-109 110-120 190 658 659 920 9211-23 24-39 40-54 55-61 62-93 94-102 103-112 1-30 31-35 36-49 50-66 67-9899-105 106-116 194 660 661 922 923 1-23 24-39 40-54 55-61 62-93 94-102103-112 1-30 31-35 36-49 50-66 67-98 99-113 114-124 197 662 663 924 9251-23 24-39 40-54 55-61 62-93 94-102 103-112 1-30 31-35 36-49 50-66 67-9899-113 114-124 198 664 665 926 927 1-23 24-39 40-54 55-61 62-93 94-102103-112 1-30 31-35 36-49 50-66 67-98 99-106 107-117 201 666 667 928 9291-23 24-39 40-54 55-61 62-93 94-102 103-112 1-30 31-35 36-49 50-66 67-9899-110 111-121 205 668 669 930 931 1-23 24-39 40-54 55-61 62-93 94-102103-112 1-30 31-35 36-49 50-66 67-98 99-113 114-124 206 670 671 932 9331-23 24-39 40-54 55-61 62-93 94-102 103-112 1-30 31-35 36-49 50-66 67-9899-112 113-123 208 None 672 None 934 — — — — — — — 1-30 31-35 36-4950-66 67-98 99-112 113-123 211 673 674 935 936 1-23 24-39 40-54 55-6162-93 94-102 103-112 1-30 31-35 36-49 50-66 67-98 99-110 111-121 251 675676 937 938 1-22 23-33 34-48 49-55 56-87 88-97   98-107 1-30 31-35 36-4950-66 67-98 99-115 116-126 252 672 673 939 940 1-22 23-33 34-48 49-5556-87 88-96   97-106 1-30 31-35 36-49 50-66 67-98 99-120 121-131 253 679680 841 942 1-22 23-33 34-48 49-55 56-87 88-96   97-106 1-30 31-35 36-4950-66 67-98 99-115 116-126 254 681 682 943 944 1-22 23-33 34-48 49-5556-87 88-96   97-106 1-30 31-35 36-49 50-66 67-98 99-115 116-126 255 683684 945 946 1-23 24-35 36-50 51-57 58-89 90-98   99-108 1-30 31-35 36-4950-66 67-98 99-114 115-125 256 685 686 947 947 1-22 23-33 34-48 49-5556-87 88-96   97-106 1-30 31-35 36-49 50-66 67-98 99-105 106-116 257 687688 949 950 1-22 23-33 34-48 49-55 56-87 88-96   97-106 1-30 31-35 36-4950-66 67-98 99-118 119-129 259 689 690 951 952 1-22 23-33 34-48 49-5556-87 88-96   97-106 1-30 31-35 36-49 50-66 67-98 99-114 115-125 260 691692 953 954 1-23 24-34 35-49 50-56 57-88 89-98   99-108 1-30 31-35 36-4950-66 67-98 99-114 115-125 261 693 694 955 956 1-23 24-35 36-50 51-5758-89 90-98   99-108 1-30 31-35 36-49 50-66 67-98 99-113 114-124 262 695696 957 958 1-23 24-34 35-49 50-56 57-88 89-98   99-108 1-30 31-35 36-4950-66 67-98 99-108 109-119 263 697 698 959 960 1-23 24-34 35-49 50-5657-88 89-98   99-108 1-30 31-35 36-49 50-66 67-98 99-112 113-123 264 699700 961 962 1-23 24-34 35-49 50-56 57-88 89-98   99-108 1-30 31-35 36-4950-66 67-98 99-109 110-120 265 701 702 963 964 1-23 24-34 35-49 50-5657-88 89-97   98-107 1-30 31-35 36-49 50-66 67-98 99-113 114-124 266 703704 965 966 1-23 24-34 35-49 50-56 57-88 89-99  100-109 1-30 31-35 36-4950-66 67-98 99-108 109-119 267 705 706 967 968 1-23 24-34 35-49 50-5657-88 89-97   98-107 1-30 31-35 36-49 50-66 67-98 99-106 107-117 270 707708 969 970 1-23 24-39 40-54 55-61 62-93 94-102 103-112 1-30 31-35 36-4950-66 67-98 99-112 113-123 271 709 710 971 972 1-23 24-39 40-54 55-6162-93 94-102 103-112 1-30 31-35 36-49 50-66 67-98 99-112 113-123 272 711721 973 974 1-23 24-39 40-54 55-61 62-93 94-102 103-112 1-30 31-35 36-4950-66 67-98 99-113 114-124 273 713 714 975 976 1-23 24-34 35-49 50-5657-88 89-97   98-107 1-30 31-35 36-49 50-66 67-98 99-105 106-116 274 715716 977 978 1-23 24-39 40-54 55-61 62-93 94-102 103-112 1-30 31-35 36-4950-66 67-98 99-112 113-123 275 717 718 979 980 1-23 24-39 40-54 55-6162-93 94-102 103-112 1-30 31-35 36-49 50-66 67-98 99-105 106-138 276 719720 981 982 1-23 24-34 35-49 50-56 57-88 89-97   98-107 1-30 31-35 36-4950-66 67-98 99-105 106-116 277 721 722 983 984 1-23 24-34 35-49 50-5657-88 89-97   98-107 1-30 31-35 36-49 50-66 67-98 99-105 106-116 278 723724 985 986 1-23 24-39 40-54 55-61 62-93 94-102 103-112 1-30 31-35 36-4950-66 67-98 99-112 113-123 279 725 726 987 988 1-22 23-36 37-51 52-5859-90 91-102 103-112 1-30 31-35 36-49 50-66 67-98 99-112 113-123 280 727728 989 990 1-23 24-39 40-54 55-61 62-93 94-102 103-112 1-30 31-35 36-4950-66 67-98 99-112 113-123 281 729 730 991 992 1-23 24-39 40-54 55-6162-93 94-102 103-112 1-30 31-35 36-49 50-66 67-98 99-112 113-123 282 731732 993 994 1-23 24-39 40-54 55-61 62-93 94-102 103-112 1-30 31-35 36-4950-66 67-98 99-112 113-123 283 733 734 995 996 1-22 23-36 37-51 52-5859-90 91-102 103-112 1-30 31-35 36-49 50-66 67-98 99-112 113-123 284 735736 997 998 1-22 23-36 37-51 52-58 59-90 91-102 103-112 1-30 31-35 36-4950-66 67-98 99-112 113-123 285 737 738 999 1000 1-23 24-39 40-54 55-6162-93 94-102 103-112 1-30 31-35 36-49 50-66 67-98 99-112 113-123 287 739740 1001 1002 1-23 24-39 40-54 55-61 62-93 94-102 103-112 1-30 31-3536-49 50-66 67-98 99-113 114-124 288 741 742 1003 1004 1-22 23-36 37-5152-58 59-90 91-102 103-112 1-30 31-35 36-49 50-66 67-98 99-112 113-123289 743 744 1005 1006 1-23 24-39 40-54 55-61 62-93 94-102 103-112 1-3031-35 36-49 50-66 67-98 99-112 113-123 290 745 None 1007 None 1-23 24-3435-49 50-56 57-88 89-97   98-107 — — — — — — — 298 None 746 None 1008 —— — — — — — 1-30 31-35 36-49 50-66 67-98 99-115 116-126 299 747 None1009 None 1-23 24-34 35-49 50-56 57-88 89-97   98-107 — — — — — — — 301749 None 1011 None 1-23 24-34 35-49 50-56 57-88 89-97   98-107 — — — — —— — 304 750 751 1012 1013 1-22 23-36 37-51 52-58 59-90 91-102 103-1121-30 31-35 36-49 50-66 67-98 99-112 113-123 305 752 753 1014 1015 1-2223-36 37-51 52-58 59-90 91-102 103-112 1-30 31-35 36-49 50-66 67-9899-112 113-123

TABLE 3 Correlation of cluster number with clones in cell-based assays:Cluster number IL-17A clones M7.19.D10 86 M7.19.F4 83 M7.20.E5 95M7.24.E8 99 M7.20.G6 97 M7.24.G6 100 M7.24.E5 98 [M7.19] E7 87 M7.24.C287 M7.20.F4 88 M7.24.A5 88 M7.24.A4 88 M7.20.C10 94 M7.20.F11 96M7.20.A9 90 M7.24.D8 87 [M7.19] E7 87 IL-23p19 clones M7.12 B9 41 M7.12F9 29 M7.9 G9 36 M7.13 D7 M7.3 D4 101 M7.9 A7 27 M7.7 F5 102 M7.12 A7103 M7.13 A6 103 M7.36.B6 305 M7.36.D3 304 M7.35.E9 303 M7.35.C9 302

The scFv entities that bind IL-17A or IL-23p19 can be oriented with thevariable light region either amino terminal to the variable heavy regionor carboxylterminal to it. Additionally, tandem scFvs can be prepared ina number of configurations, such that each target, i.e, IL-17A andIL-23p19 can be bound by its respective variable regions. Thus, theconstruct for a tandem scFV molecule can be prepared such that thevariable light region and variable heavy region of one antibody can beinterspersed with the variable light and variable heavy regions of theother antibody as long as the variable regions are able to bind thetargets. Tandem scFv molecules that bind both targets can be preparedwith a linker between the scFv entities, including a Gly-Ser linkercomprising a series of glycine and serine residues and can also includeadditional amino acids. Tandem bispecific scFv molecules with thevariable regions of cluster numbers c103 and c87 exhibited an IC50 (nM)of 4.3 in the assay described in Example 5 herein.

The antibodies and derivatives thereof of the invention have bindingaffinities that include a dissociation constant (K_(d)) of less than1×10⁻². In some embodiments, the K_(d) is less than 1×10⁻³. In otherembodiments, the K_(d) is less than 1×10⁻⁴. In some embodiments, theK_(d) is less than 1×10⁻⁵. In still other embodiments, the K_(d) is lessthan 1×10⁻⁶. In other embodiments, the K_(d) is less than 1×10⁻⁷. Inother embodiments, the K_(d) is less than lx 10⁻⁸. In other embodiments,the K_(d) is less than 1×10⁻⁹. In other embodiments, the K_(d) is lessthan 1×10⁻¹⁰. In still other embodiments, the K_(d) is less than1×10⁻¹¹. In some embodiments, the K_(d) is less than 1×10⁻¹². In otherembodiments, the K_(d) is less than 1×10⁻¹³. In other embodiments, theK_(d) is less than 1×10⁻¹⁴. In still other embodiments, the K_(d) isless than 1×10⁻¹⁵.

The anti-IL-17A and anti-IL23p19 antibodies isolated and describedherein have been grouped into families of consensus CDRs, which areshown in SEQ ID NOs: 31 to 77 for anti-IL-17A and SEQ ID NOs: 326 to 491for anti-IL-23p19. Table 4, below correlates the SEQ ID NOs: with theanti-IL-17A consensus CDRs.

TABLE 4 CDR family LCDR1 LCDR2 LCDR3 HCDR1 HCDR2 HCDR3 A 31 32 33 34 3536-37 B 38 39 40 41 42 43 C 44 45 46 47 48 49-50 D 51 52 53 54 55 56-61E 62 63 64 65 66 67 F 68 69 70 71 72 37, 57, 73-77

Table 5, below correlates the SEQ ID NOs: with the anti-IL-23p19consensus CDRs.

TABLE 5 CDR family LCDR1 LCDR2 LCDR3 HCDR1 HCDR2 HCDR3 A 326 327 328 329330 331-340 B 341 342 343 344 345 331, 346-352 C 353 354 355 356 357358-364 D 365 366 367 368 369 370-372 E 373 374 375 376 377 378-404 F405 406 407 408 409 410-411 G 412 413 414 415 416 417-420 H 421 422 423424 425 426-429 I 430 431 432 433 434 435-450 J 451 452 453 453 455456-458 K 459 460 461 462 463 464-466 L 467 468 469 470 471 472-475 M476 477 478 479 480 481-490

Thus, the invention provides an antibody that binds a polypeptidecomprising IL-17A (SEQ ID NO:2), wherein the antibody comprises: a lightchain variable region comprising: i) a light chain CDR1 selected fromthe group consisting of: 1) SEQ ID NO: 31; 2) SEQ ID NO: 38; 3) SEQ IDNO: 44; 4) SEQ ID NO: 51; 5) SEQ ID NO: 62; and 6) SEQ ID NO: 68; andii) a light chain CDR2 selected from the group consisting of: 1) SEQ IDNO: 32; 2) SEQ ID NO: 39; 3) SEQ ID NO: 45; 4) SEQ ID NO:52; 5) SEQ IDNO: 63; and 6) SEQ ID NO: 69; and iii) a light chain CDR3 selected fromthe group consisting of: 1) SEQ ID NO: 33; 2) SEQ ID NO: 40; 3) SEQ IDNO: 46; 4) SEQ ID NO: 53; 5) SEQ ID NO: 64; and 6) SEQ ID NO: 70; and b)a heavy chain variable region comprising: i) a heavy chain CDR1 selectedfrom the group consisting of: 1) SEQ ID NO: 34; 2) SEQ ID NO: 41; 3) SEQID NO: 47; 4) SEQ ID NO: 54; 5) SEQ ID NO: 65; and 6) SEQ ID NO: 71; andii) a heavy chain CDR2 selected from the group consisting of: 1) SEQ IDNO: 35; 2) SEQ ID NO: 42; 3) SEQ ID NO: 48; 4) SEQ ID NO: 55; 5) SEQ IDNO: 66; and 6) SEQ ID NO: 72; and iii) a heavy chain CDR3 selected fromthe group consisting of: 1) SEQ ID NO: 36-37; 2) SEQ ID NO: 43; 3) SEQID NO: 49-50; 4) SEQ ID NO: 56-61; 5) SEQ ID NO: 67; and 6) SEQ ID NO:37, 57, and 73-77. Within an embodiment, the antibody is an antibodyfragment. Within another embodiment the antibody is selected from thegroups consisting of: Fv, Fab, Fab′, F(ab)₂, F(ab′)₂, scFv, and diabody.Within another embodiment the antibody is a bispecific molecule. Withinanother embodiment the antibody also binds IL-23p19 (SEQ ID NO: 4).

The invention provides an antibody that binds a polypeptide comprisingSEQ ID NO:2, wherein the antibody is selected from the group consistingof: a) the antibody comprising the light chain CDR1 of SEQ ID NO: 31,the light chain CDR2 of SEQ ID NO: 32, the light chain CDR3 of SEQ IDNO: 33, the heavy chain CDR1 of SEQ ID NO:34, the heavy chain CDR2 ofSEQ ID NO: 35, and wherein the heavy chain CDR3 is selected from SEQ IDNOs: 36-37; b) the antibody comprising the light chain CDR1 of SEQ IDNO: 38, the light chain CDR2 of SEQ ID NO: 39, the light chain CDR3 ofSEQ ID NO: 40, the heavy chain CDR1 of SEQ ID NO: 41, the heavy chainCDR2 of SEQ ID NO: 42, and wherein the heavy chain CDR3 is SEQ ID NOs:43; c) the antibody comprising the light chain CDR1 of SEQ ID NO: 44,the light chain CDR2 of SEQ ID NO: 45, the light chain CDR3 of SEQ IDNO:46, the heavy chain. CDR1 of SEQ ID NO: 47, the heavy chain CDR2 ofSEQ ID NO: 48, and wherein the heavy chain CDR3 is selected from SEQ IDNOs: 49-50; d) the antibody comprising the light chain CDR1 of SEQ II)NO: 51, the light chain CDR2 of SEQ ID NO: 52, the light chain CDR3 ofSEQ ID NO: 53, the heavy chain CDR1 of SEQ ID NO: 54, the heavy chain.CDR2 of SEQ ID NO: 55, and wherein the heavy chain CDR3 is selected fromSEQ ID NOs: 56-61; e) the antibody comprising the light chain CDR1 ofSEQ ID NO: 62, the light chain CDR2 of SEQ ID NO: 63, the light chainCDR3 of SEQ ID NO: 64, the heavy chain CDR1 of SEQ ID NO: 65, the heavychain CDR2 of SEQ ID NO: 66, and wherein the heavy chain CDR3 is SEQ IDNOs: 67; and f) the antibody comprising the light chain CDR1 of SEQ IDNO: 68, the light chain CDR2 of SEQ ID NO: 69, the light chain CDR3 ofSEQ ID NO: 70, the heavy chain CDR1 of SEQ ID NO:71, the heavy chainCDR2 of SEQ ID NO: 72, and wherein the heavy chain CDR3 is selected fromSEQ ID NOs: 37, 57, or 73-77.

The invention provides an antibody that binds a polypeptide comprisingIL-23 (SEQ ID NO:4), wherein the antibody comprises: i) a light chainCDR1 selected from the group consisting of 1) SEQ ID NO: 326; 2) SEQ IDNO: 341; 3) SEQ ID NO: 353; 4) SEQ ID NO: 365; 5) SEQ ID NO: 373: 6) SEQID NO: 405; 7) SEQ ID NO: 412; 8) SEQ ID NO: 421; 9) SEQ ID NO: 430; 10)SEQ ID NO: 451; 11) SEQ ID NO: 459; 12) SEQ ID NO: 467; and 13) SEQ IDNO: 476; and ii) a light chain CDR2 selected from the group consistingof: 1) SEQ ID NO: 327; 2) SEQ ID NO: 342; 3) SEQ ID NO: 354; 4) SEQ IDNO: 366; 5) SEQ ID NO: 374; 6) SEQ ID NO: 406; 7) SEQ ID NO: 413; 8) SEQID NO: 422; 9) SEQ ID NO: 431; 10) SEQ ID NO: 452; 11) SEQ ID NO: 460;12) SEQ ID NO: 468; and 13) SEQ ID NO: 477; and iii) a light chain CDR3selected from the group consisting of: 1) SEQ ID NO: 328; 2) SEQ ID NO:343; 3) SEQ ID NO: 355; 4) SEQ ID NO: 367; 5) SEQ ID NO: 375; 6) SEQ IDNO: 407; 7) SEQ ID NO: 414; 8) SEQ ID NO: 423; 9) SEQ ID NO: 432; 10)SEQ ID NO: 453; 11) SEQ ID NO: 461; 12) SEQ ID NO: 469; 13) SEQ ID NO:478; and b) a heavy chain variable region comprising: i) a heavy chainCDR1 selected from the group consisting of: 1) SEQ ID NO: 329; 2) SEQ IDNO: 344; 3) SEQ ID NO: 356; 5) SEQ ID NO: 368; 5) SEQ ID NO: 376; 6) SEQID NO: 408; 7) SEQ ID NO: 415; 8) SEQ ID NO: 424; 9) SEQ ID NO: 433; 10)SEQ ID NO: 453; 11) SEQ ID NO: 462; 12) SEQ ID NO: 470; and 13) SEQ IDNO: 479; and ii) a heavy chain CDR2 selected from the group consistingof: 1) SEQ ID NO: 330; 2) SEQ ID NO: 345; 3) SEQ ID NO: 357; 4) SEQ IDNO: 369; 5) SEQ ID NO: 377; 6) SEQ ID NO: 409; 7) SEQ ID NO: 416; 8) SEQID NO: 425; 9) SEQ ID NO: 434; 10) SEQ ID NO: 455; 11) SEQ ID NO: 463;12) SEQ ID NO: 471; and 13) SEQ ID NO: 480; and iii) a heavy chain CDR3selected from the group consisting of: 1) SEQ ID NOs: 331-340; 2) SEQ IDNOs: 331, 346-352; 3) SEQ ID NOs: 358-364; 4) SEQ ID NOs: 370-372; 5)SEQ ID NOs: 378-404; 6) SEQ ID NOs: 410-411; 7) SEQ ID NOs: 417-420; 8)SEQ ID NOs: 426-429; 9) SEQ ID NOs: 435-450; 10) SEQ ID NOs: 456-458;11) SEQ ID NOs: 464-466; 12) SEQ ID NOs: 472-475; and 13) SEQ ID NOs:481-490. Within embodiments, the antibody is an antibody fragment, Fv,Fab, Fab′, F(ab)₂, F(ab′)₂, say, and diabody, and as bispecific moleculethat binds IL-17A (SEQ ID NO: 2).

The invention provides an antibody selected from the group consistingof: a) the antibody comprising the light chain CDR1 of SEQ ID NO: 326,the light chain CDR2 of SEQ ID NO: 327, the light chain CDR3 of SEQ IDNO: 328, the heavy chain CDR1 of SEQ ID NO:329, the heavy chain CDR2 ofSEQ ID NO: 330, and wherein the heavy chain CDR3 is selected from SEQ IDNOs: 331-340; b) the antibody comprising the light chain CDR1 of SEQ IDNO: 341, the light chain CDR2 of SEQ ID NO: 342, the light chain CDR3 ofSEQ ID NO: 343, the heavy chain CDR1 of SEQ ID NO: 344, the heavy chainCDR2 of SEQ ID NO: 345 and wherein the heavy chain CDR3 is SEQ ID NOs:331, and 346-352; c) the antibody comprising the light chain CDR1 of SEQID NO: 353, the light chain CDR2 of SEQ ID NO: 354, the light chain CDR3of SEQ ID NO:355, the heavy chain CDR1 of SEQ ID NO: 356, the heavychain CDR2 of SEQ ID NO: 357, and wherein the heavy chain CDR3 isselected from SEQ ID NOs: 358-364; d) the antibody comprising the lightchain CDR1 of SEQ ID NO: 365, the light chain CDR2 of SEQ ID NO: 366,the light chain CDR3 of SEQ ID NO: 367, the heavy chain CDR1 of SEQ IDNO: 368, the heavy chain CDR2 of SEQ ID NO: 369, and wherein the heavychain CDR3 is selected from SEQ ID NOs: 370-372; e) the antibodycomprising the light chain CDR1 of SEQ ID NO: 373, the light chain CDR2of SEQ ID NO: 374, the light chain CDR3 of SEQ ID NO: 375, the heavychain CDR1 of SEQ ID NO: 376, the heavy chain CDR2 of SEQ ID NO: 377,and wherein the heavy chain CDR3 is SEQ ID NOs: 378-04; f) the antibodycomprising the light chain CDR1 of SEQ ID NO: 405, the light chain CDR2of SEQ ID NO: 406, the light chain CDR3 of SEQ ID NO: 407, the heavychain CDR1 of SEQ ID NO:408, the heavy chain CDR2 of SEQ ID NO: 409, andwherein the heavy chain CDR3 is selected from SEQ ID NOs: 37, 57, or410-411; g) the antibody comprising the light chain CDR1 of SEQ ID NO:412, the light chain CDR2 of SEQ ID NO: 413, the light chain CDR3 of SEQID NO: 414, the heavy chain CDR1 of SEQ ID NO: 415, the heavy chain CDR2of SEQ ID NO: 416, and wherein the heavy chain CDR3 is selected from SEQID NOs: 417-420; h) the antibody comprising the light chain CDR1 of SEQID NO: 421, the light chain CDR2 of SEQ ID NO: 422, the light chain.CDR3 of SEQ ID NO: 423, the heavy chain CDR1 of SEQ ID NO:424, the heavychain CDR2 of SEQ ID NO: 425, and wherein the heavy chain CDR3 isselected from SEQ ID NOs: 426-429; i) the antibody comprising the lightchain CDR1 of SEQ ID NO: 430, the light chain CDR2 of SEQ ID NO: 431,the light chain CDR3 of SEQ ID NO: 432, the heavy chain CDR1 of SEQ IDNO: 433, the heavy chain CDR2 of SEQ ID NO: 434, and wherein the heavychain CDR3 is selected from SEQ ID NOs: 435-450; j) the antibodycomprising the light chain CDR1 of SEQ ID NO: 451, the light chain CDR2of SEQ ID NO: 452, the light chain CDR3 of SEQ ID NO: 453, the heavychain CDR1 of SEQ ID NO:454, the heavy chain CDR2 of SEQ ID NO: 455, andwherein the heavy chain CDR3 is selected from SEQ ID NOs: 456-458; k)the antibody comprising the light chain CDR1 of SEQ ID NO: 459, thelight chain CDR2 of SEQ ID NO: 460, the light chain CDR3 of SEQ ID NO:461, the heavy chain CDR1 of SEQ ID NO:462, the heavy chain CDR2 of SEQID NO: 463, and wherein the heavy chain CDR3 is selected from SEQ IDNOs:464-466; 1) the antibody comprising the light chain CDR1 of SEQ IDNO: 467, the light chain CDR2 of SEQ ID NO: 468, the light chain CDR3 ofSEQ ID NO: 469, the heavy chain CDR1 of SEQ ID NO:470, the heavy chainCDR2 of SEQ ID NO: 471, and wherein the heavy chain CDR3 is selectedfrom SEQ ID NOs: 472-475; and m) the antibody comprising the light chainCDR1 of SEQ ID NO: 476, the light chain CDR2 of SEQ ID NO: 477, thelight chain CDR3 of SEQ ID NO: 478, the heavy chain CDR1 of SEQ IDNO:479, the heavy chain CDR2 of SEQ ID NO: 480, and wherein the heavychain CDR3 is selected from SEQ ID NOs: 481-490. Within embodiments, theantibody is an antibody fragment, Fv, Fab, Fab′, F(ab)₂, F(ab′)₂, scFv,and diabody, and as bispecific molecule that binds IL-17A (SEQ ID NO:2).

The invention provides antibodies that binds polypeptide comprising SEQID NO:2, wherein the portion of SEQ ID NO: 2 where the antibody binds isamino acid residues 77 to 85 of SEQ ID NO: 2. Antibodies to epitopesfrom other mammalian species of IL-17 can also be useful. As such, theinvention provides antibodies that bind to binds a polypeptidecomprising 1) SEQ ID NO:1022 (cynomolgus monkey), wherein the portion ofSEQ ID NO: 1022 where the antibody binds is selected from the groupconsisting of: a) amino acid residues 77 to 85 of SEQ ID NO: 1022; andb) amino acid residues 123 to 128 of SEQ ID NO: 1022; 2) SEQ ID NO:1023(murine), wherein the portion of SEQ ID NO: 1023 where the antibodybinds is selected from the group consisting of a) amino acid residues 80to 88 of SEQ ID NO: 1023; and b) amino acid residues 125 to 131 of SEQID NO: 1023; and c) SEQ ID NO:1024 (rat), wherein the portion of SEQ IDNO: 1024 where the antibody binds is selected from the group consistingof: a) amino acid residues 72 to 80 of SEQ ID NO: 1024; and b) aminoacid residues 117 to 123 of SEQ ID NO: 1024.

The invention provides antibodies that neutralize the polypeptidecomprising SEQ ID NO:2, wherein the portion of SEQ ID NO: 2 where theantibody binds is selected from the group consisting of a) amino acidresidues 34 to 41 of SEQ ID NO: 2; and b) amino acid residues 52 to 64of SEQ ID NO: 2. Antibodies to epitopes from other mammalian species ofIL-17 can also be useful. As such, the invention provides antibodiesthat bind to binds a polypeptide comprising 1) SEQ ID NO: 1022(cynomologus monkey) where the antibody binds is selected from the groupconsisting of: a) amino acid residues 34 to 41 of SEQ ID NO: 1022; andb) amino acid residues 52 to 64 of SEQ ID NO: 1022; 2) SEQ ID NO: 1023(murine) where the antibody binds is selected from the group consistingof: a) amino acid residues 36 to 43 of SEQ ID NO: 1023; and b) aminoacid residues 60 to 67 of SEQ ID NO: 1023; 3) SEQ ID NO: 1024 (rat)where the antibody binds is amino acid residues 49 to 59 of SEQ ID NO:1024.

The invention provides antibodies that binds a polypeptide comprisingSEQ ID NO:4, wherein the portion of SEQ ID NO: 4 where the antibodybinds is selected from the group consisting of: a) amino acid residues55 to 66 of SEQ ID NO: 4; and b) amino acid residues 74 to 85 of SEQ IDNO: 4. Antibodies to epitopes from other mammalian species of IL-17 canalso be useful. As such, the invention provides antibodies that bind tobinds a polypeptide comprising 1) SEQ ID NO: 1025 (cyno) where theantibody binds is selected from the group consisting of: a) amino acidresidues 55 to 66 of SEQ ID NO: 1025; and b) amino acid residues 74 to85 of SEQ ID NO: 1025; 2) SEQ ID NO: 1026 (murine) where the antibodybinds is selected from the group consisting of: a) amino acid residues56 to 67 of SEQ ID NO: 1026; and b) amino acid residues 73-86 of SEQ IDNO: 1026; and 3) SEQ ID NO: 1027 (rat) where the antibody binds isselected from the group consisting of: a) amino acid residues 55 to 68of SEQ ID NO: 1027; and b) amino acid residues 73 to 86 of SEQ ID NO:1027.

The invention provides antibodies that neutralize the polypeptidecomprising SEQ ID NO:2, wherein the portion of SEQ ID NO: 4 where theantibody binds is selected from the group consisting of: a) amino acidresidues 137 to 146 of SEQ ID NO: 4; and b) amino acid residues 155 to164 of SEQ ID NO: 4. Antibodies to epitopes from other mammalian speciesof IL-17 can also be useful. As such, the invention provides antibodiesthat bind to binds a polypeptide comprising 1) SEQ ID NO: 1025 (cyno)where the antibody binds is selected from the group consisting of: a)amino acid residues 137 to 146 of SEQ ID NO: 1025; and b) amino acidresidues 155 to 164 of SEQ ID NO: 1025; 2) SEQ ID NO: 1026 (murine)where the antibody binds is selected from the group consisting of: a)amino acid residues 137 to 146 of SEQ ID NO: 1026; and b) amino acidresidues 155 to 165 of SEQ ID NO: 1026; and 3) where the antibody bindsis selected from the group consisting of: a) amino acid residues 137 to147 of SEQ ID NO: 1027; and b) amino acid residues 155 to 165 of SEQ IDNO: 1027.

The invention provides an antibody that binds a polypeptide comprisingIL-17A (SEQ ID NO: 2) comprising a light chain variable region selectedfrom the group consisting of: SEQ ID NO: 80, 84, 88, 92, 96, 100, 104,108, 112, 116, 120, 124, 128, 132, 136, 140, 144, 148, 152, 156, 160,164, 168, 172, 176, 180, 184, 188, 192, 196, 200, 204, 208, 212, 216,220, 224, 228, 232, 236, 240, 244, 248, 252, 256, 260, 264, 268, 272,276, 280, 284, 288, 292, 296, 300, 304, 308, 312, 316, 320, and 325.Within an embodiment, the antibody further comprising a heavy chainvariable region selected from the group consisting of: SEQ ID NO: 81,85, 89, 93, 97, 101, 105, 109, 113, 117, 121, 125, 129, 133, 137, 141,145, 149, 153, 157, 161, 165, 169, 173, 177, 181, 185, 189, 193, 197,201, 205, 209, 213, 217, 221, 225, 229, 233, 237, 241, 245, 249, 253,257, 261, 265, 269, 273, 277, 281, 285, 289, 293, 297, 301, 305, 309,313, 317, 321, and 323. Within an embodiment, the light chain variableregion is amino terminus to the heavy chain variable region or the heavychain variable region is amino terminal to the light chain variableregion. Within an embodiment, the antibody is an antibody fragment.Within an embodiment, the antibody is selected from the group consistingof: Fv, Fab, Fab′, F(ab)₂, F(ab′)₂, scFv, and diabody. Within anembodiment, the antibody is a bispecific molecule. Within an embodiment,the bispecific molecule also binds IL-23p19 (SEQ ID NO: 4).

The invention provides an antibody that binds a polypeptide comprisingSEQ ID NO: 2 comprising a heavy chain variable region selected from thegroup consisting of: SEQ ID NO: 81, 85, 89, 93, 97, 101, 105, 109, 113,117, 121, 125, 129, 133, 137, 141, 145, 149, 153, 157, 161, 165, 169,173, 177, 181, 185, 189, 193, 197, 201, 205; 209, 213, 217, 221, 225,229, 233, 237, 241, 245, 249, 253, 257, 261, 265, 269, 273, 277, 281,285, 289, 293, 297, 301, 305, 309, 313, 317, 321, and 323. Within anembodiment, the antibody further comprising a light chain variableregion selected from the group consisting of: SEQ ID NO: 80, 84, 88, 92,96, 100, 104, 108, 112, 116, 120, 124, 128, 132, 136, 140, 144, 148,152, 156, 160, 164, 168, 172, 176, 180, 184, 188, 192, 196, 200, 204,208, 212, 216, 220, 224, 228, 232, 236, 240, 244, 248, 252, 256, 260,264, 268, 272, 276, 280, 284, 288, 292, 296, 300, 304, 308, 312, 316,320, and 325. Within an embodiment, the light chain variable region isamino terminus to the heavy chain variable region or the heavy chainvariable region is amino terminal to the light chain variable region.Within an embodiment, the antibody is an antibody fragment, Within anembodiment, the antibody is selected from the groups consisting of: Fv,Fab, Fab′, F(ab)₂, F(ab′)₂, scFv, and diabody. Within an embodiment, theantibody is a bispecific molecule. Within an embodiment, the bispecificmolecule also binds IL-23p19 (SEQ ID NO: 4).

The invention provides an antibody that binds a polypeptide comprisingSEQ ID NO: 2 comprising a light chain variable region selected from thegroup consisting of: SEQ ID NO: 92, 80, 128, 144, 136, 148, 140, 96,100, 124, 132, and 108 and comprising a heavy chain variable regionselected from the group consisting of: SEQ ID NO: 93, 81, 129, 145, 137,149, 141, 97, 101, 125, 133, and 109. Within an embodiment, the antibodyis an antibody fragment. Within an embodiment, antibody is selected fromthe groups consisting of: Fv, Fab, Fab′, F(ab)₂, F(ab′)₂, scFv, anddiabody. Within an embodiment, is a bispecific molecule. Within anembodiment, bispecific molecule also binds IL-23p19 (SEQ ID NO: 4).

The invention provides an antibody that binds a polypeptide comprisingSEQ ID NO: 2, wherein the antibody comprises a light chain and a heavychain selected from the group consisting of: a) a light chain comprisingSEQ ID NO: 92 and a heavy chain comprising SEQ ID NO: 93; b) a lightchain comprising SEQ ID NO: 80 and a heavy chain comprising SEQ ID NO:81; c) a light chain comprising SEQ ID NO: 128 and a heavy chaincomprising SEQ ID NO: 129; d) a light chain comprising SEQ ID NO: 144and a heavy chain comprising SEQ ID NO: 145; e) a light chain comprisingSEQ ID NO: 136 and a heavy chain comprising SEQ ID NO: 137; f) a lightchain comprising SEQ ID NO: 148 and a heavy chain comprising SEQ ID NO:149; g) a light chain comprising SEQ ID NO: 140 and a heavy chaincomprising SEQ ID NO: 141; h) a light chain comprising SEQ ID NO: 96 anda heavy chain comprising SEQ ID NO: 97; i) a light chain comprising SEQID NO: 100 and a heavy chain comprising SEQ ID NO: 101; j) a light chaincomprising SEQ ID NO: 124 and a heavy chain comprising SEQ ID NO: 125;k) a light chain comprising SEQ ID NO: 132 and a heavy chain comprisingSEQ ID NO: 133; and) a light chain comprising SEQ ID NO: 108 and a heavychain comprising SEQ ID NO: 109. Within an embodiment, the antibody isan antibody fragment. Within an embodiment, the antibody is selectedfrom the groups consisting of Fv, Fab, Fab′, F(ab)₂, F(ab′)₂, scFv, anddiabody. Within an embodiment, the antibody is a bispecific molecule.that also binds IL-23p19 (SEQ ID NO: 4). Within an embodiment, the lightchain variable region is amino terminal or carboxyl terminal to theheavy chain variable region.

The invention provides an antibody that binds a polypeptide comprisingSEQ ID NO: 2, wherein the antibody competes for binding with an antibodycomprising a light chain and a heavy chain selected from the groupconsisting of: a) a light chain comprising SEQ ID NO: 92 and a heavychain comprising SEQ ID NO: 93; b) a light chain comprising SEQ ID NO:80 and a heavy chain comprising SEQ ID NO: 81; c) a light chaincomprising SEQ ID NO: 128 and a heavy chain comprising SEQ ID NO: 129;d) a light chain comprising SEQ ID NO: 144 and a heavy chain comprisingSEQ ID NO: 145; e) a light chain comprising SEQ ED NO: 136 and a heavychain comprising SEQ ID NO: 137; t) a light chain comprising SEQ ID NO:96 and a heavy chain comprising SEQ ID NO: 97; g) a light chaincomprising SEQ ID NO: 100 and a heavy chain comprising SEQ ID NO: 101;h) a light chain comprising SEQ ID NO: 124 and a heavy chain comprisingSEQ ID NO: 125; i) a light chain comprising SEQ ID NO: 132 and a heavychain comprising SEQ ID NO: 133; a j) a light chain comprising SEQ IDNO: 108 and a heavy chain comprising SEQ ID NO: 109. Within embodiments,the antibody is an antibody fragment, Fv, Fab, Fab′, F(ab)₂, F(ab′)₂,scFv, and diabody, a bispecific molecule that also binds IL-23p19 (SEQID NO: 4).

The invention provides an antibody that binds a polypeptide comprisingSEQ ID NO: 2, wherein the antibody competes for binding with an antibodycomprising a light chain comprising SEQ ID NO: 148 and a heavy chaincomprising SEQ ID NO: 149. Within embodiments, the antibody is anantibody fragment, Fv, Fab, Fab′, F(ab)₂, F(ab′)₂, scFv, and diabody, abispecific molecule that also binds IL-23p19 (SEQ ID NO: 4).

The invention provides an antibody that binds a polypeptide comprisingSEQ ID NO: 4 comprising a light chain variable region selected from thegroup consisting of: SEQ ID NO: 754, 756, 758, 460, 762, 764, 766, 768,770, 772, 774, 776, 778, 780, 782, 784, 786, 788, 790, 792, 794, 796,798, 800, 802, 804, 806, 808, 810, 812, 814, 816, 818, 820, 822, 824,826, 828, 830, 832, 834, 836, 838, 840, 842, 844, 846, 848, 850, 825,854, 856, 858, 860, 862, 864, 866, 868, 870, 872, 874, 876, 878, 880,882, 884, 886, 888, 890, 892, 894, 896, 898, 902, 904, 906, 908, 910,912, 914, 916, 918, 920, 922, 924, 926, 928, 930, 932, 935, 937, 939,941, 943, 945, 947, 949, 951, 953, 955, 957, 959, 961, 963, 965, 967,969, 971, 973, 975, 977, 979, 981, 983, 985, 987, 989, 991, 993, 995,997, 999, 1001, 1003, 1005, 1007, 1010, 1011, 1012, and 1014. Within anembodiment, the antibody further comprising a heavy chain variableregion selected form the group consisting of: 755, 757, 759, 761, 763,765, 767, 769, 771, 773, 775, 777, 779, 781, 783, 785, 787, 789, 791,793, 795, 797, 799, 801, 803, 805, 807, 809, 811, 813, 815, 817, 819,820, 823, 825, 827, 829, 831, 833, 835, 837, 839, 841, 843, 845, 847,849, 851, 853, 855, 857, 859, 861, 863, 865, 867, 869, 871, 873, 875,877, 879, 881, 883, 885, 887, 889, 891, 893, 895, 897, 899, 903, 905,907, 909, 911, 913, 915, 917, 919, 921, 923, 925, 927, 929, 931, 933,934, 936, 938, 940, 942, 944, 946, 947, 950, 952, 954, 956, 958, 960,962, 964, 966, 968, 970, 972, 974, 976, 978, 980, 982, 984, 986, 988,990, 992, 994, 996, 998, 1000, 1002, 1004, 1006, 1008, 1013, and 1015.Within embodiments the antibody is an antibody fragment, is selectedfrom the groups consisting of: Fv, Fab, Fab′, F(ab)₂, F(ab′)₂, scFv, anddiabody, is a bispecifie molecule that binds IL-17A (SEQ ID NO: 2).

The invention provides an antibody that binds a polypeptide comprisingSEQ ID NO: 4 comprising a light chain variable region selected from thegroup consisting of: SEQ ID NO: 768 and 764 and comprising a heavy chainvariable region selected from the group consisting of: SEQ ID NO: 769and 765. Within embodiments the antibody is an antibody fragment, isselected from the groups consisting of: Fv, Fab, Fab′, F(ab)₂, F(ab′)₂,scFv, and diabody, is a bispecific molecule that binds IL-17A (SEQ IDNO: 2).

The invention provides an antibody that binds a polypeptide comprisingSEQ ID NO: 4 comprising a light chain variable region selected from thegroup consisting of: SEQ ID NO: 774 and 776 and comprising a heavy chainvariable region selected from the group consisting of: SEQ ID NO: 775and 777. Within embodiments the antibody is an antibody fragment, isselected from the groups consisting of: Fv, Fab, Fab′, F(ab)₂, F(ab′)₂,scFv, and diabody, is a bispecific molecule that binds IL-17A (SEQ IDNO: 2).

The invention provides methods of treating autoimmune diseases includingmultiple sclerosis, IBD, and demyelinating diseases. The inventionprovides, a method for inhibiting interleukin-17 (IL-17) production by Tcells comprising treating said T cells with an antagonist of IL-17 andIL-23, wherein said IL-23 antagonist binds the p19 subunit of IL-23. TheT cells may be activated and may be memory cells.

The invention provides methods of treating diseases characterized byelevated expression of IL-17 or IL-23 in a mammalian subject, comprisingadministering to said subject an effective amount of an antagonist ofIL-17 and IL-23, including diseases such as multiple sclerosis (MS),chronic inflammation, autoimmune diabetes, rheumatoid arthritis (RA) andother arthritic conditions, asthma, systhemic lupus erythrematosus,psoriasis, Crohn's Disease, ulcerative colitis, irritable bowel syndrome(IBS) and inflammatory bowel disease (IBD). Within embodiments theantibody is an antibody fragment, is selected from the groups consistingof: Fv, Fab, Fab′, F(ab)₂, F(ab′)₂, scFv, and diabody, is a bispecificmolecule that binds IL-17A (SEQ ID NO: 2) and IL-23p19 (SEQ ID NO: 4).

The invention provides a method for preventing, inhibiting, or reducingrelapse in multiple sclerosis comprising administering a combination ofan antagonist to IL-17A and an antagonist to IL-23p19. In an embodiment,the antagonist to IL-17A and the antagonist to IL-23p19 areco-administered. In another embodiment, the antagonist to IL-17A is ascFv molecule and the antagonist to IL-23p19 is a scFv molecule. Inanother embodiment, the antagonist to IL-17A and an antagonist toIL-23p19 are administered in one entity. Within an embodiment, theentity is a bispecific molecule. Within an embodiment, the antagonist toIL-17A comprises a light chain selected from the group consisting of:SEQ ID NO: 80, 84, 88, 92, 96, 100, 104, 108, 112, 116, 120, 124, 128,132, 136, 140, 144, 148, 152, 156, 160, 164, 168, 172, 176, 180, 184,188, 192, 196, 200, 204, 208, 212, 216, 220, 224, 228, 232, 236, 240,244, 248, 252, 256, 260, 264, 268, 272, 276, 280, 284, 288, 292, 296,300, 304, 308, 312, 316, 320, and 325, and/or comprises a heavy chainselected from the group consisting of: SEQ ID NO: 81, 85, 89, 93, 97,101, 105, 109, 113, 117, 121, 125, 129, 133, 137, 141, 145, 149, 153,157, 161, 165, 169, 173, 177, 181, 185, 189, 193, 197, 201, 205, 209,213, 217, 221, 225, 229, 233, 237, 241, 245, 249, 253, 257, 261, 265,269, 273, 277, 281, 285, 289, 293, 297, 301, 305, 309, 313, 317, 321,and 323. Within an embodiment, the antagonist to IL-23p19 comprises alight chain selected from the group consisting of: SEQ ID NO: SEQ ID NO:754, 756, 758, 460, 762, 764, 766, 768, 770, 772, 774, 776, 778, 780,782, 784, 786, 788, 790, 792, 794, 796, 798, 800, 802, 804, 806, 808,810, 812, 814, 816, 818, 820, 822, 824, 826, 828, 830, 832, 834, 836,838, 840, 842, 844, 846, 848, 850, 825, 854, 856, 858, 860, 862, 864,866, 868, 870, 872, 874, 876, 878, 880, 882, 884, 886, 888, 890, 892,894, 896, 898, 902, 904, 906, 908, 910, 912, 914, 916, 918, 920, 922,924, 926, 928, 930, 932, 935, 937, 939, 941, 943, 945, 947, 949, 951,953, 955, 957, 959, 961, 963, 965, 967, 969, 971, 973, 975, 977, 979,981, 983, 985, 987, 989, 991, 993, 995, 997, 999, 1001, 1003, 1005,1007, 1010, 1011, 1012, and 1014, and/or comprising a heavy chainvariable region selected form the group consisting of: SEQ ID NO: 755,757, 759, 761, 763, 765, 767, 769, 771, 773, 775, 777, 779, 781, 783,785, 787, 789, 791, 793, 795, 797, 799, 801, 803, 805, 807, 809, 811,813, 815, 817, 819, 820, 823, 825, 827, 829, 831, 833, 835, 837, 839,841, 843, 845, 847, 849, 851, 853, 855, 857, 859, 861, 863, 865, 867,869, 871, 873, 875, 877, 879, 881, 883, 885, 887, 889, 891, 893, 895,897, 899, 903, 905, 907, 909, 911, 913, 915, 917, 919, 921, 923, 925,927, 929, 931, 933, 934, 936, 938, 940, 942, 944, 946, 947, 950, 952,954, 956, 958, 960, 962, 964, 966, 968, 970, 972, 974, 976, 978, 980,982, 984, 986, 988, 990, 992, 994, 996, 998, 1000, 1002, 1004, 1006,1008, 1013, and 1015. Similarly, the invention provides, a method forpreventing, inhibiting, or reducing IBD comprising administering acombination of an antagonist to IL-17A and an antagonist to IL-23p19.

The invention provides an isolated antibody that binds both IL-17A (SEQID NO: 2) and IL-23p19 (SEQ ID NO: 4) comprising at least one of thefollowing: a) a light chain variable region selected from the groupconsisting of: SEQ ID NO: 80, 84, 88, 92, 96, 100, 104, 108, 112, 116,120, 124, 128, 132, 136, 140, 144, 148, 152, 156, 160, 164, 168, 172,176, 180, 184, 188, 192, 196, 200, 204, 208, 212, 216, 220, 224, 228,232, 236, 240, 244, 248, 252, 256, 260, 264, 268, 272, 276, 280, 284,288, 292, 296, 300, 304, 308, 312, 316, 320, and 325; b) a heavy chainvariable region selected from the group consisting of: SEQ ID NO: 81,85, 89, 93, 97, 101, 105, 109, 113, 117, 121, 125, 129, 133, 137, 141,145, 149, 153, 157, 161, 165, 169, 173, 177, 181, 185, 189, 193, 197,201, 205, 209, 213, 217, 221, 225, 229, 233, 237, 241, 245, 249, 253,257, 261, 265, 269, 273, 277, 281, 285, 289, 293, 297, 301, 305, 309,313, 317, 321, and 323; c) a light chain variable region selected fromthe group consisting of: SEQ ID NO: 754, 756, 758, 460, 762, 764, 766,768, 770, 772, 774, 776, 778, 780, 782, 784, 786, 788, 790, 792, 794,796, 798, 800, 802, 804, 806, 808, 810, 812, 814, 816, 818, 820, 822,824, 826, 828, 830, 832, 834, 836, 838, 840, 842, 844, 846, 848, 850,825, 854, 856, 858, 860, 862, 864, 866, 868, 870, 872, 874, 876, 878,880, 882, 884, 886, 888, 890, 892, 894, 896, 898, 902, 904, 906, 908,910, 912, 914, 916, 918, 920, 922, 924, 926, 928, 930, 932, 935, 937,939, 941, 943, 945, 947, 949, 951, 953, 955, 957, 959, 961, 963, 965,967, 969, 971, 973, 975, 977, 979, 981, 983, 985, 987, 989, 991, 993,995, 997, 999, 1001, 1003, 1005, 1007, 1010, 1011, 1012, and 1014; andd) a heavy chain variable region selected from the group consisting of:SEQ ID NO: 755, 757, 759, 761, 763, 765, 767, 769, 771, 773, 775, 777,779, 781, 783, 785, 787, 789, 791, 793, 795, 797, 799, 801, 803, 805,807, 809, 811, 813, 815, 817, 819, 820, 823, 825, 827, 829, 831, 833,835, 837, 839, 841, 843, 845, 847, 849, 851, 853, 855, 857, 859, 861,863, 865, 867, 869, 871, 873, 875, 877, 879, 881, 883, 885, 887, 889,891, 893, 895, 897, 899, 903, 905, 907, 909, 911, 913, 915, 917, 919,921, 923, 925, 927, 929, 931, 933, 934, 936, 938, 940, 942, 944, 946,947, 950, 952, 954, 956, 958, 960, 962, 964, 966, 968, 970, 972, 974,976, 978, 980, 982, 984, 986, 988, 990, 992, 994, 996, 998, 1000, 1002,1004, 1006, 1008, 1013, and 1015.

D) Nucleic Acids

The invention also includes nucleic acids encoding the heavy chainand/or light chain of the antibodies of the invention. Nucleic acids ofthe invention include nucleic acids having at least 80%, more preferablyat least about 90%, more preferably at least about 95%, and mostpreferably at least about 98% homology to nucleic acids of theinvention. The terms “percent similarity”, “percent identity” and“percent homology” when referring to a particular sequence are used asset forth in the University of Wisconsin GCG software program. Nucleicacids of the invention also include complementary nucleic acids. In someinstances, the sequences will be fully complementary (no mismatches)when aligned. In other instances, there may be up to about a 20%mismatch in the sequences. In some embodiments of the invention areprovided nucleic acids encoding both a heavy chain and a light chain ofan antibody of the invention. One skilled in the art will know that thenucleic acid sequences provided herein can be exploited using codonoptimization, degenerate sequence, silent mutations, and other DNAtechniques to optimize expression in a particular hosts. The inventionencompasses such sequence modifications. The present invention providespolynucleotide molecules, including DNA and RNA molecules, that encodethe anti-IL-17A and anti-IL-23p19 antibodies disclosed herein. Thoseskilled in the art will readily recognize that, in view of thedegeneracy of the genetic code, considerable sequence variation ispossible among these polynucleotide molecules.

Nucleic acids of the invention can be cloned into a vector, such as aplasmid, cosmid, bacmid, phage, artificial chromosome (BAC, YAC) orvirus, into which another genetic sequence or element (either DNA orRNA) may be inserted so as to bring about the replication of theattached sequence or element. In some embodiments, the expression vectorcontains a constitutively active promoter segment (such as but notlimited to CMV, SV40, Elongation Factor or LTR sequences) or aninducible promoter sequence such as the steroid inducible pIND vector(Invitrogen), where the expression of the nucleic acid can be regulated.Expression vectors of the invention may further comprise regulatorysequences, for example, an internal ribosomal entry site. The expressionvector can be introduced into a cell by transfection, for example.

E) Methods of Producing Antibodies to IL-17 and IL-23

The invention also provides methods of producing monoclonal antibodiesthat specifically bind to IL-17 and IL-23, either singly or together.Antibodies of the invention may be produced in vivo or in vitro. Onestrategy for generating antibodies against both IL-17 and IL-23 involvesimmunizing animals with both IL-17 and IL-23. In some embodiments,animals are immunized with the monomeric or multimeric form of oth IL-17and IL-23. Animals so immunized will produce antibodies against bothIL-17 and IL-23. Standard methods are known for creating monoclonalantibodies including, but are not limited to, the hybridoma technique(see Kohler & Milstein, (1975) Nature 256:495-497); the triomatechnique; the human B-cell hybridoma technique (see Kozbor et al.(1983) Immunol. Today 4:72) and the EBV hybridoma technique to producehuman monoclonal antibodies (see Cole, et al. in MONOCLONAL ANTIBODIESAND CANCER THERAPY, Alan R. Liss, Inc., 1985, pp. 77-96).

Both IL-17 and IL-23 may be purified from cells or from recombinantsystems using a variety of well-known techniques for isolating andpurifying proteins. For example, but not by way of limitation, bothIL-17 and IL-23 may be isolated based on the apparent molecular weightof the protein by running the protein on an SDS-PAGE gel and blottingthe proteins onto a membrane. Thereafter, the appropriate size bandcorresponding to either protein may be cut from the membrane and used asan immunogen in animals directly, or by first extracting or eluting theprotein from the membrane. As an alternative example, the protein may beisolated by size-exclusion chromatography alone or in combination withother means of isolation and purification.

The invention also provides methods of producing monoclonal antibodiesthat specifically bind to homodimeric, heterodimeric, and/or multimericforms of both IL-17 and IL-23/p19. These different forms may be purifiedfrom cells or from recombinant systems using a variety of well-knowntechniques for isolating and purifying proteins. For example, but not byway of limitation, both IL-17 and IL-23/p19 may be isolated based on theapparent molecular weight of the protein by running the protein on anSDS-PAGE gel and blotting the proteins onto a membrane. Thereafter, theappropriate size band corresponding to each may be cut from the membraneand used as an immunogen in animals directly, or by first extracting oreluting the protein from the membrane. As an alternative example, theprotein may be isolated by size-exclusion chromatography alone or incombination with other means of isolation and purification.

Other means of purification are available in such standard referencetexts as Zola, Monoclonal Antibodies: Preparation And Use Of MonoclonalAntibodies And Engineered Antibody Derivatives (Basics: From BackgroundTo Bench) Springer-Verlag Ltd., New York, 2000; Basic Methods InAntibody Production And Characterization, Chapter 11, “AntibodyPurification Methods,” Howard and Bethell, Eds., CRC Press, 2000;Antibody Engineering (Springer Lab Manual.), Kontermann and Dubel, Eds.,Springer-Verlag, 200

For in vivo antibody production, animals are generally immunized witheither IL-17 or IL-23 or an immunogenic portion of either. The antigenis generally combined with an adjuvant to promote immunogenicity.Adjuvants vary according to the species used for immunization. Examplesof adjuvants include, but are not limited to: Complete Freund's Adjuvant(“CFA”), Incomplete Freund's Adjuvant (“IFA”), mineral gels (e.g.,aluminum hydroxide), surface active substances (e.g., lysolecithin,pluronic polyols, polyanions), peptides, oil emulsions, keyhole limpethemocyanin (“KLH”), dinitrophenol (“DNP”), and potentially useful humanadjuvants such as Bacille Calmette-Guerin (“BCG”) and corynebacteriumparvum. Such adjuvants are also well known in the art. Immunization maybe accomplished using well-known procedures. The dose and immunizationregimen will depend on the species of mammal immunized, its immunestatus, body weight, and/or calculated surface area, etc. Typically,blood serum is sampled from the immunized mammals and assayed foranti-IL-17 and IL-23/p19 antibodies using appropriate screening assaysas described below, for example.

A common method for producing humanized antibodies is to graft CDRsequences from a MAb (produced by immunizing a rodent host) onto a humanIg backbone, and transfection of the chimeric genes into Chinese HamsterOvary (CHO) cells which in turn produce a functional Ab that is secretedby the CHO cells (Shields, R. L., et al. (1995) Int Arch. AllergyImmunol. 107:412-413). The methods described within this application arealso useful for generating genetic alterations within Ig genes orchimeric Igs transfected within host cells such as rodent cell lines,plants, yeast and prokaryotes (Frigerio L, et al. (2000) Plant Physiol.123:1483-1494).

Splenocytes from immunized animals may be immortalized by fusing thesplenocytes (containing the antibody-producing B cells) with an immortalcell line such as a myeloma line. Typically, myeloma cell line is fromthe same species as the splenocyte donor. In one embodiment, theimmortal cell line is sensitive to culture medium containinghypoxanthine, aminopterin and thymidine (“HAT medium”). In someembodiments, the myeloma cells are negative for Epstein-Barr virus (EBV)infection. In preferred embodiments, the myeloma cells areHAT-sensitive, EBV negative and Ig expression negative. Any suitablemyeloma may be used. Murine hybridomas may be generated using mousemyeloma cell lines (e.g., the P3-NS1/1-Ag4-1, P3-x63-Ag8.653 orSp2/O-Ag14 myeloma lines). These murine myeloma lines are available fromthe ATCC. These myeloma cells are fused to the donor splenocytespolyethylene glycol (“PEG”), preferably 1500 molecular weightpolyethylene glycol (“PEG 1500”). Hybridoma cells resulting from thefusion are selected in HAT medium which kills unfused and unproductivelyfused myeloma cells. Unfused splenocytes die over a short period of timein culture. In some embodiments, the myeloma cells do not expressimmunoglobulin genes.

Hybridomas producing a desired antibody which are detected by screeningassays such as those described below may be used to produce antibodiesin culture or in animals. For example, the hybridoma cells may becultured in a nutrient medium under conditions and for a time sufficientto allow the hybridoma cells to secrete the monoclonal antibodies intothe culture medium. These techniques and culture media are well known bythose skilled in the art. Alternatively, the hybridoma cells may beinjected into the peritoneum of an unimmunized animal. The cellsproliferate in the peritoneal cavity and secrete the antibody, whichaccumulates as ascites fluid. The ascites fluid may be withdrawn fromthe peritoneal cavity with a syringe as a rich source of the monoclonalantibody.

Another non-limiting method for producing human antibodies is describedin U.S. Pat. No. 5,789,650 which describes transgenic mammals thatproduce antibodies of another species (e.g., humans) with their ownendogenous immunoglobulin genes being inactivated, The genes for theheterologous antibodies are encoded by human immunoglobulin genes. Thetransgenes containing the unrearranged immunoglobulin encoding regionsare introduced into a non-human animal. The resulting transgenic animalsare capable of functionally rearranging the transgenic immunoglobulinsequences and producing a repertoire of antibodies of various isotypesencoded by human immunoglobulin genes. The B-cells from the transgenicanimals are subsequently immortalized by any of a variety of methods,including fusion with an immortalizing cell line (e.g., a myeloma cell).

The antibodies of the present invention may also be prepared in vitrousing a variety of techniques known in the art. For example, but not byway of limitation, fully human monoclonal antibodies against IL-17 andIL-23/p19 may be prepared by using in vitro-primed human splenocytes(Boerner et al. (1991) J. Immunol. 147:86-95).

Alternatively, for example, the antibodies of the invention may beprepared by “repertoire cloning” (Persson et al. (1991) Proc. Nat. Acad.Sci. USA 88:2432-2436; and Huang and Stollar (1991) J. Immunol. Methods141:227-236). Further, U.S. Pat. No. 5,798,230 describes preparation ofhuman monoclonal antibodies from human B antibody-producing B cells thatare immortalized by infection with an Epstein-Barr virus that expressesEpstein-Barr virus nuclear antigen 2 (EBNA2). EBNA2, required forimmortalization, is then inactivated resulting in increased antibodytiters.

In another embodiment, antibodies of the invention are formed by invitro immunization of peripheral blood mononuclear cells (“PBMCs”). Thismay be accomplished by any means known in the art, such as, for example,using methods described in the literature (Zafiropoulos et al. (1997)Immunological Methods 200:181-190).

In a specific embodiment, bispecific and single chain antibodies thatbind both IL-17 and IL-23 are made. One method of the present inventionis a method for producing a bispecific IL-17/IL-23 antibody. The methodcomprises fusing hybridoma cells that secrete a monoclonal antibody thatbinds IL-17, with hybridoma cells that secrete a monoclonal antibodythat binds IL-23/p19, thereby preparing a hybrid hybridoma that secretesa bispecific IL-17/IL-23 monoclonal antibody. In one embodiment, themethod comprises fusing hybridoma cells that secrete an antagonistic (oragonistic) IL-17 MAb, with hybridoma cells that secrete an antagonistic(or agonistic) IL-23/p19 MAb. Conventional techniques for conductingsuch a fusion, and for isolating the desired hybrid hybridoma, includethose described elsewhere herein, and those illustrated in the examplesbelow.

U.S. Pat. No. 6,060,285 discloses a process for the production ofbispecific antibodies, in which at least the genes for the light chainand the variable portion of the heavy chain of an antibody having afirst specificity are transfected into a hybridoma cell secreting anantibody having a second specificity. When the transfected hybridomacells are cultured, bispecific antibodies are produced, and may beisolated by various means known in the art.

Other investigators have used chemical coupling of antibody fragments toprepare antigen-binding molecules having specificity for two differentantigens (Brennan et al., Science 229:81 1985; Glennie et al., J.Immunol. 139:2367, 1987). U.S. Pat. No. 6,010,902 also discussestechniques known in the art by which bispecific antibodies can beprepared, for example by the use of heterobifunctional cross-linkingreagents such as GMBS (maleimidobutyryloxy succinimide) or SPDP(N-succinimidyl 3-(2-pyridyldithio)propionate). (See, e.g., Hardy,“Purification And Coupling Of Fluorescent Proteins For Use In FlowCytometry”, Handbook Of Experimental Immunology, 4.sup.th Ed., Volume 1,Immunochemistry, Weir et al. (eds.), pp. 31.4-31.12, 1986).

The ability to produce antibodies via recombinant DNA technology hasfacilitated production of bispecific antibodies. Kostelny et al.utilized the leucine zipper moieties from the fos and jun proteins(which preferentially form heterodimers) to produce bispecificantibodies able to bind both the cell surface molecule CD3 and thereceptor for IL-2 (J. Immunol. 148:1547; 1992).

Single chain antibodies may be formed by linking heavy and light chainvariable region (Fv region) fragments via an amino acid bridge (shortpeptide linker), resulting in a single polypeptide chain. Suchsingle-chain Fvs (scFvs) have been prepared by fusing DNA encoding apeptide linker between DNAs encoding the two variable regionpolypeptides (V.sub.L and V.sub.H). The resulting antibody fragments canform dimers or higher oligomers, depending on such factors as the lengthof a flexible linker between the two variable domains (Kortt et al.,Protein Engineering 10:423, 1997). In particular embodiments, two ormore scFvs are joined by use of a chemical cross-linking agent.

Techniques developed for the production of single chain antibodies canbe adapted to produce single chain antibodies of the present invention,that bind both IL-17 and IL-23. Such techniques include those describedin U.S. Pat. No. 4,946,778; Bird (Science 242:423, 1988); Huston et al.(Proc. Natl. Acad. Sci. USA 85:5879, 1988); and Ward et al. (Nature334:544, 1989). Once desired single chain antibodies are identified (forexample, from a phage-display library), those of skill in the art canfurther manipulate the DNA encoding the single chain antibody(ies) toyield bispecific antibodies, including bispecific antibodies having Fcregions.

Single chain antibodies against IL-17 and IL-23 may be concatamerized ineither order (i.e., anti-IL-17-anti-IL-23 or anti-IL-23-anti-IL-17). Inparticular embodiments, starting materials for preparing a bispecificIL-17/IL-23 antibody include an antagonistic (or agonistic) single chainantibody directed against IL-17 and an antagonistic (or agonistic)single chain antibody directed against IL-23/p19.

U.S. Pat. No. 5,582,996 discloses the use of complementary interactivedomains (such as leucine zipper moieties or other lock and keyinteractive domain structures) to facilitate heterodimer formation inthe production of bispecific antibodies. The complementary interactivedomain(s) may be inserted between an Fab fragment and another portion ofa heavy chain (i.e., C.sub.H1 or C.sub.H2 regions of the heavy chain).The use of two different Fab fragments and complementary interactivedomains that preferentially heterodimerize will result in bispecificantibody molecules. Cysteine residues may be introduced into thecomplementary interactive domains to allow disulphide bonding betweenthe complementary interactive domains and stabilize the resultingbispecific antibodies.

Tetravalent, bispecific molecules can be prepared by fusion of DNAencoding the heavy chain of an F(ab′).sub.2 fragment of an antibody witheither DNA encoding the heavy chain of a second F(ab).sub.2 molecule (inwhich the CH1 domain is replaced by a CH3 domain), or with DNA encodinga single chain Fv fragment of an antibody, as described in U.S. Pat. No.5,959,083. Expression of the resultant fusion genes in mammalian cells,together with the genes for the corresponding light chains, yieldstetravalent bispecific molecules having specificity for selectedantigens.

Bispecific antibodies can also be produced as described in U.S. Pat. No.5,807,706, which is incorporated by reference herein. Generally, themethod involves introducing a protuberance in a first polypeptide and acorresponding cavity in a second polypeptide, polypeptides interface.The protuberance and cavity are positioned so as to promoteheteromultimer formation and hinder homomultimer formation. Theprotuberance is created by replacing amino acids having small sidechains with amino acids having larger side chains. The cavity is createdby the opposite approach, i.e., replacing amino acids having relativelylarge side chains with amino acids having smaller side chains.

The protuberance and cavity can be generated by conventional methods formaking amino acid substitutions in polypeptides. For example, a nucleicacid encoding a polypeptide may be altered by conventional in vitromutagenesis techniques. Alternatively, a polypeptide incorporating adesired amino acid substitution may be prepared by peptide synthesis.Amino acids chosen for substitution are located at the interface betweenthe first and second polypeptides.

F) Screening for Antibody Specificity

Screening for antibodies that specifically bind to IL-17 and/orIL-23/p19 may be accomplished using the procedures and assays known inthe art and those described herein. For example, an enzyme-linkedimmunosorbent assay (ELISA) can be used in which microtiter plates arecoated with either or both IL-17 and IL-23 (or p19 alone). In someembodiments, antibodies that bind both IL-17 and IL-23/p19 frompositively reacting clones can be further screened for reactivity in anELISA-based assay using microtiter plates coated with the other formsIL-17 and IL-23/p19, or other IL-17 family members. Clones that produceantibodies that are reactive to another forms or family members areeliminated, and clones that produce antibodies that are reactive to bothIL-17 and IL-23/p19 may be selected for further expansion anddevelopment. Confirmation of reactivity of the antibodies to both IL-17and IL-23/p19 may be accomplished, for example, using a Western Blotassay in which protein from ovarian, breast, renal, colorectal, lung,endometrial, or brain cancer cells and purified FR-.alpha. and otherfolate receptor isoforms are run on an SDS-PAGE gel, and subsequentlyare blotted onto a membrane. The membrane may then be probed with theputative anti-FR-.alpha. antibodies. Reactivity with both IL-17 andIL-23/p19 and not another family member confirms specificity ofreactivity for both IL-17 and IL-23/p19.

In some embodiments, the binding affinity of the antibodies of thepresent invention antibodies is determined. Antibodies of the inventionpreferably have a binding affinity to IL-17 and IL-23/p19 (either singlyor together as with a bispecific antibody or scFV) of at least about1.times.10.sup.−7 M, more preferably at least about 1.times.10.sup.−8 M,more preferably at least about 1.times.10.sup.−9 M, and most preferablyat least about 1.times.10.sup.−10 M. Preferred antibody-producing cellsof the invention produce substantially only antibodies having a bindingaffinity to IL-17 and IL-23/p19 (either singly or together as with abispecific antibody or scFV) of at least about 1.times.10.sup.−7 M, morepreferably at least about 1.times.10.sup.−8 M, more preferably at leastabout 1.times.10.sup.−9 M, and most preferably at least about1.times.10.sup.−10 M. Preferred compositions of the invention comprisesubstantially only antibodies having a binding affinity to both IL-17and IL-23/p19 (either singly or together as with a bispecific antibodyor scFV) of at least about 1.times.10.sup.−7 M, more preferably at leastabout 1.times.10.sup.−8 M, more preferably at least about1.times.10.sup.−9 M, and most preferably at least about1.times.10.sup.−10 M.

G) Anti-IL-17 and IL-23/p19 Antibody-Producing Cells

Antibody-producing cells of the invention include any insect expressioncell line known, such as for example, Spodoptera frugiperda cells. Theexpression cell lines may also be yeast cell lines, such as, forexample, Saccharomyces cerevisiae and Schizosaccharomyces pombe cells.The expression cells may also be mammalian cells such as, for example,hybridoma cells (e.g., NS0 cells), Chinese hamster ovary cells, babyhamster kidney cells, human embryonic kidney line 293, normal dog kidneycell lines, normal cat kidney cell lines, monkey kidney cells, Africangreen monkey kidney cells, COS cells, and non-tumorigenic mouse myoblastG8 cells, fibroblast cell lines, myeloma cell lines, mouse NIH/3T3cells, LMTK31 cells, mouse sertoli cells, human cervical carcinomacells, buffalo rat liver cells, human lung cells, human liver cells,mouse mammary tumor cells, TRI cells, MRC 5 cells, and FS4 cells.

In some preferred embodiments, the antibody-producing cells of theinvention produce antibodies that specifically bind to IL-17 andIL-23/p19 (either singly or together as with a bispecific antibody orscFV). The cells preferably are substantially free of both IL-17 andIL-23 binding competitors. In preferred embodiments, theantibody-producing cells comprise less than about 10%, preferably lessthan about 5%, more preferably less than about 1%, more preferably lessthan about 0.5%, more preferably less than about 0.1%, and mostpreferably 0% by weight both IL-17 and IL-23 binding competitors. Insome preferred embodiments, the antibodies produced by theantibody-producing cells are substantially free of both IL-17 and IL-23competitors. In preferred embodiments, antibodies produced by theantibody-producing cells comprise less than about 10%, preferably lessthan about 5%, more preferably less than about 1%, more preferably lessthan about 0.5%, more preferably less than about 0.1%, and mostpreferably 0% by weight both IL-17 and IL-23 binding competitors.Preferred antibody-producing cells of the invention producesubstantially only antibodies having a binding affinity to IL-17 andIL-23/p19 (either singly or together as with a bispecific antibody orscFV) of at least about 1.times.10.sup.−7 M, more preferably at leastabout 1.times.10.sup.−8 M, more preferably at least about1.times.10.sup.−9 M, and most preferably at least about1.times.10.sup.−10 M.

H) Antibody Purification

Methods of antibody purification are known in the art. In someembodiments of the invention, methods for antibody purification includefiltration, affinity column chromatography, cation exchangechromatography, anion exchange chromatography, and concentration. Thefiltration step preferably comprises ultrafiltration, and morepreferably ultrafiltration and diafiltration. Filtration is preferablyperformed at least about 5-50 times, more preferably 10 to 30 times, andmost preferably 14 to 27 times. Affinity column chromatography, may beperformed using, for example, PROSEP Affinity Chromatography (Millipore,Billerica, Mass.). In a preferred embodiment, the affinitychromatography step comprises PROSEP-VA column chromatography. Eluatemay be washed in a solvent detergent. Cation exchange chromatography mayinclude, for example, SP-Sepharose Cation Exchange Chromatography. Anionexchange chromatography may include, for example but not limited to,Q-Sepharose Fast Flow Anion Exchange. The anion exchange step ispreferably non-binding, thereby allowing removal of contaminantsincluding DNA and BSA. The antibody product is preferably nanofiltered,for example, using a Pall DV 20 Nanofilter. The antibody product may beconcentrated, for example, using ultrafiltration and diafiltration. Themethod may further comprise a step of size exclusion chromatography toremove aggregates.

I) Therapeutic Uses of the IL-17 and IL-23/p19 Antibodies

Antibodies that bind to both IL-17 and IL-23 can be used to modulate theimmune system by binding IL-17 and IL-23/p19 (either singly or togetheras with a bispecific antibody or scFV), and thus, preventing the bindingof IL-17 with either IL-17RA and/or IL-17RC and IL-23 with its receptor(IL-12RB1/IL-23R) or any other receptor that they may bind, especiallyan IL-17 receptor family member. The antibodies of the invention canalso be used to modulate the immune system by inhibiting the binding ofboth IL-17 with the endogenous IL-17RA and/or IL-17RC receptor and IL-23with its endogenous receptor (IL-12RB1/IL-23R). The antibodies of theinvention can be also used to treat a subject which produces an excessof either IL-17 and/or IL-23. Suitable subjects include mammals, such ashumans. For example, the antibodies of the invention are useful inbinding, blocking, inhibiting, reducing, antagonizing or neutralizing ofboth IL-17 and IL-23 (either singly or together as with a bispecificantibody or scFV), in the treatment of inflammation and inflammatorydiseases such as multiple sclerosis, demyelinating diseases, autoimmuneocular disease, uveitis, scleritis, cancer (characterized by IL-17 andIL-23 expression), psoriasis, IBS, inflammatory bowel disease (IBD),colitis, promotion of tumor growth, arthritis, or degenerative jointdisease and other inflammatory conditions disclosed herein.

Within preferred embodiments, the antibodies of the invention bind to,blocks, inhibits, reduces, antagonizes or neutralizes IL-23 (via p19)and IL-17 either singly or together (as with a bispecific antibody orscFV), in vivo.

Moreover, the antibodies of the invention are useful to:

(1) Block, inhibit, reduce, antagonize or neutralize signaling via IL-17and IL-23 in the treatment of cancer, acute inflammation, and chronicinflammatory diseases such as inflammatory bowel disease (IBD), IBS,chronic colitis, splenomegaly, rheumatoid arthritis, and other diseasesassociated with the induction of acute-phase response.

(2) Block, inhibit, reduce, antagonize or neutralize signaling via IL-17or IL-23 in the treatment of autoimmune diseases such asinsulin-dependent diabetes mellitus (IDDM), multiple sclerosis (MS),demyelinating diseases, autoimmune ocular disease, uveitis, scleritis,systemic Lupus erythematosus (SLE), myasthenia gravis, rheumatoidarthritis, IBS and IBD to prevent or inhibit signaling in immune cells(e.g. lymphocytes, monocytes, leukocytes) via their receptors (e.g.IL-17RA and IL-17RC). Blocking, inhibiting, reducing, or antagonizingsignaling via IL-17RA and IL-17RC, using the antibodies of the presentinvention, may also benefit diseases of the pancreas, kidney, pituitaryand neuronal cells. IDDM, non-insulin dependent diabetes mellitus(NIDDM), pancreatitis, and pancreatic carcinoma may benefit.

The antibodies described herein can be used to bind, block, inhibit,reduce, antagonize or neutralize IL-23 and IL-17 activity, either singlyor together as with a bispecific antibody or scFV, in the treatment ofmultiple sclerosis, cancer, autoimmune disease, atopic disease, NIDDM,pancreatitis and kidney dysfunction as described above. The antibodiesof the present invention are useful as antagonists of IL-17 or IL-23.Such antagonistic effects can be achieved by direct neutralization orbinding of IL-17 and IL-23 (via p19).

Antibodies herein can also be directly or indirectly conjugated todrugs, toxins, radionuclides and the like, and these conjugates used forin vivo diagnostic or therapeutic applications. For instance, antibodiesor binding polypeptides which recognize IL-17 or IL-23 can be used toidentify or treat tissues or organs that express a correspondinganti-complementary molecule. More specifically, antibodies to IL-17 orIL-23 or bioactive fragments or portions thereof, can be coupled todetectable or cytotoxic molecules and delivered to a mammal havingcells, tissues or organs that express these cytokines IL-17 orIL-23-expressing cancers.

Suitable detectable molecules may be directly or indirectly attached tothe antagonists of the present invention, such as “bindingpolypeptides,” (including binding peptides disclosed above), antibodies,or bioactive fragments or portions thereof. Suitable detectablemolecules include radionuclides, enzymes, substrates, cofactors,inhibitors, fluorescent markers, chemiluminescent markers, magneticparticles and the like. Suitable cytotoxic molecules may be directly orindirectly attached to the polypeptide or antibody, and includebacterial or plant toxins (for instance, diphtheria toxin, Pseudomonasexotoxin, ricin, abrin and the like), as well as therapeuticradionuclides, such as iodine-131, rhenium-188 or yttrium-90 (eitherdirectly attached to the polypeptide or antibody, or indirectly attachedthrough means of a chelating moiety, for instance). Binding polypeptidesor antibodies may also be conjugated to cytotoxic drugs, such asadriamycin. For indirect attachment of a detectable or cytotoxicmolecule, the detectable or cytotoxic molecule can be conjugated with amember of a complementary/anticomplementary pair, where the other memberis bound to the binding polypeptide or antibody portion. For thesepurposes, biotin/streptavidin is an exemplarycomplementary/anticomplementary pair.

In another embodiment, binding polypeptide-toxin fusion proteins orantibody-toxin fusion proteins can be used for targeted cell or tissueinhibition or ablation (for instance, to treat cancer cells or tissues).Alternatively, if the binding polypeptide has multiple functionaldomains (i.e., an activation domain or a ligand binding domain, plus atargeting domain), a fusion protein including only the targeting domainmay be suitable for directing a detectable molecule, a cytotoxicmolecule or a complementary molecule to a cell or tissue type ofinterest. In instances where the fusion protein including only a singledomain includes a complementary molecule, the anti-complementarymolecule can be conjugated to a detectable or cytotoxic molecule. Suchdomain-complementary molecule fusion proteins thus represent a generictargeting vehicle for cell/tissue-specific delivery of genericanti-complementary-detectable/cytotoxic molecule conjugates.

Inflammation is a protective response by an organism to fend off aninvading agent. Inflammation is a cascading event that involves manycellular and humoral mediators. On one hand, suppression of inflammatoryresponses can leave a host immunocompromised; however, if leftunchecked, inflammation can lead to serious complications includingchronic inflammatory diseases (e.g., asthma, psoriasis, arthritis,rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease andthe like), septic shock and multiple organ failure. Importantly, thesediverse disease states share common inflammatory mediators. Thecollective diseases that are characterized by inflammation have a largeimpact on human morbidity and mortality. Therefore it is clear thatanti-inflammatory proteins, such as antagonists to IL-17 and IL-23/p19,such as IL-17 and IL-23/p19 antibodies, could have crucial therapeuticpotential for a vast number of human and animal diseases, from asthmaand allergy to autoimmunity, cancers, and septic shock.

1. Arthritis

Arthritis, including osteoarthritis, rheumatoid arthritis, arthriticjoints as a result of injury, and the like, are common inflammatoryconditions which would benefit from the therapeutic use ofanti-inflammatory proteins, such as the antagonists of the presentinvention. For example, rheumatoid arthritis (RA) is a systemic diseasethat affects the entire body and is one of the most common forms ofarthritis. It is characterized by the inflammation of the membranelining the joint, which causes pain, stiffness, warmth, redness andswelling. Inflammatory cells release enzymes that may digest bone andcartilage. As a result of rheumatoid arthritis, the inflamed jointlining, the synovium, can invade and damage bone and cartilage leadingto joint deterioration and severe pain amongst other physiologiceffects. The involved joint can lose its shape and alignment, resultingin pain and loss of movement.

Rheumatoid arthritis (RA) is an immune-mediated disease particularlycharacterized by inflammation and subsequent tissue damage leading tosevere disability and increased mortality. A variety of cytokines areproduced locally in the rheumatoid joints. Numerous studies havedemonstrated that IL-1 and TNF-alpha, two prototypic pro-inflammatorycytokines, play an important role in the mechanisms involved in synovialinflammation and in progressive joint destruction. Indeed, theadministration of TNF-alpha and IL-1 inhibitors in patients with RA hasled to a dramatic improvement of clinical and biological signs ofinflammation and a reduction of radiological signs of bone erosion andcartilage destruction. However, despite these encouraging results, asignificant percentage of patients do not respond to these agents,suggesting that other mediators are also involved in the pathophysiologyof arthritis (Gabay, Expert. Opin. Biol. Ther. 2(2):135-149 (2002). Oneof those mediators could be IL-17 or IL-23, as demonstrated in severalreports to play a role in rheumatoid arthritis. For example, IL-17 andIL-23/p19 are overexpressed in the synovium and synovial fibroblasts ofpatients with rheumatoid arthritis compared to individuals withoutrheumatoid arthritis. Furthermore, IL-17 and IL-23/p19 have beendemonstrated to promote matrix degradation and enhance the expression ofinflammatory, matrix-destructive cytokines when added tosynovium/synoviocyte cultures. (Murphy et al, J. Exp. Med 198:1951(2003); reviewed in Lubberts et al, Arthritis Res Ther. 7:29 (2005) andKim et al, Rheumatology, 46:57 (2007)). Therefore, such a molecule thatbinds or inhibits IL-17 or IL-23 activity, such as the antagonists ofthe present invention, could serve as a valuable therapeutic to reduceinflammation in rheumatoid arthritis, and other arthritic diseases.

There are several animal models for rheumatoid arthritis known in theart. For example, in the collagen-induced arthritis (CIA) model, micedevelop chronic inflammatory arthritis that closely resembleshuman-rheumatoid arthritis. Since CIA shares similar immunological andpathological features with RA, this makes it an ideal model forscreening potential human anti-inflammatory compounds. The CIA model isa well-known model in mice that depends on both an immune response, andan inflammatory response, in order to occur. The immune responsecomprises the interaction of B-cells and CD4+ T-cells in response tocollagen, which is given as antigen, and leads to the production ofanti-collagen antibodies. The inflammatory phase is the result of tissueresponses from mediators of inflammation, as a consequence of some ofthese antibodies cross-reacting to the mouse's native collagen andactivating the complement cascade. An advantage in using the CIA modelis that the basic mechanisms of pathogenesis are known. The relevantT-cell and B-cell epitopes on type II collagen have been identified, andvarious immunological (e.g., delayed-type hypersensitivity andanti-collagen antibody) and inflammatory (e.g., cytokines, chemokines,and matrix-degrading enzymes) parameters relating to immune-mediatedarthritis have been determined, and can thus be used to assess testcompound efficacy in the CIA model (Wooley, Curr. Opin. Rheum. 3:407-20(1999); Williams et al., Immunol. 89:9784-788 (1992); Myers et al., LifeSci. 61:1861-78 (1997); and Wang et al., Immunol. 92:8955-959 (1995)).

One group has shown that an anti-mouse IL-17 antibody reduces symptomsin a mouse CIA-model relative to control mice, and another group hasshown that deficiency of IL-23/p19 is protective in CIA (Murphy et al,J. Exp. Med 198:1951 (2003)), thus showing conceptually that antagonistsof the present invention may be beneficial in treating human disease.The administration of a single mouse-IL-17-specific rat antisera reducedthe symptoms of arthritis in the animals when introducedprophylactically or after symptoms of arthritis were already present inthe model (Lubberts et al, Arthritis Rheum. 50:650-9 (2004)).

As described in the Examples below, both IL-17 and IL-23/p19 areoverexpressed in CIA. Therefore, antagonists of the present inventioncan be used to neutralize IL-17 and/or IL-23 (via p19) in the treatmentof specific human diseases such as arthritis, psoriasis, psoriaticarthritis, endotoxemia, inflammatory bowel disease (IBD), IBS, colitis,and other inflammatory conditions disclosed herein.

The administration of antagonists of the present invention to these CIAmodel mice is used to evaluate the use of these antagonists toameliorate symptoms and alter the course of disease. Moreover, resultsshowing inhibition of IL-17 and/or IL-23 signalling by these antagonistswould provide proof of concept that IL-17 and IL-23/p19 antagonists,such as those disclosed herein, can also be used to ameliorate symptomsand alter the course of disease. By way of example and withoutlimitation, the injection of 10-200 ug of an anti-IL-17 andanti-IL-23/p19 per mouse (one to seven times a week for up to but notlimited to 4 weeks via s.c., i.p., or i.m route of administration) cansignificantly reduce the disease score (paw score, incident ofinflammation, or disease). Depending on the initiation of administration(e.g. prior to or at the time of collagen immunization, or at any timepoint following the second collagen immunization, including those timepoints at which the disease has already progressed), antagonists of thepresent invention can be efficacious in preventing rheumatoid arthritis,as well as preventing its progression.

2. Inflammatory Bowel Disease IBD

In the United States approximately 1.35 million in the US (more than 1.9million in the G7 nations) have Inflammatory Bowel Disease (IBD) whichcan affect either colon and rectum (Ulcerative colitis) or both, smalland large intestine (Crohn's Disease). In both Crohn's disease andulcerative colitis, the tissue damage results from an inappropriate orexaggerated immune response to antigens of the gut microflora. Despitehaving a common basis in overresponsiveness to luminal antigens, Crohn'sdisease and ulcerative colitis are immunologically distinct entities.Crohn's disease is more associated with a Th1 T cell-mediated response,characterized by enhanced production of interferon-[gamma] and tumornecrosis factor-[alpha]. Interleukin (IL)-12 and, possibly, IL-23 governthe Th1 cell differentiation, but optimal induction and stabilization ofpolarized Th1 cells would require additional cytokines, such as IL-15,IL-18 and IL-21. In ulcerative colitis, the local immune response isless polarized, but it is characterized by CD1-reactive natural killer Tcell production of IL-13. Beyond these differences, Crohn's disease andulcerative colitis share important end-stage effector pathways ofintestinal injury, which are mediated by an active cross-talk betweenimmune and non-immune mucosal cells. As shown in the Examples andreferences below, IL-17 and IL-23 are both overexpressed in intestinesand/or serum from humans with IBD and in mouse models of IBD. (Nielsonet al, Scand J Gastroenterol. 38:180 (2003); Schmidt et al, Inflamm.Bowel Dis. 11:16 (2005); Fuss et al, Inflamm Bowel Dis. 12:9 (2006)).Moreover, neutralization of IL-17 and/or IL-23/p19 reduces diseasesymptoms and pathology in animals models of IBD (Yen et al, J. Clin.Invest. 116:1310 (2006); Zhang et al, Inflamm Bowel Dis. 12:382 (2006)).

As shown in the Examples below, both IL-17 and IL-23/p19 expression areincreased in the DSS colitis model and in the T cell transfer colitismodel, and treatment wth a combination of an anti-IL-17A antibody and ananti-IL23p19 antibody is more efficacious in the oxazalone IBD modelthan treatment with either antibody alone. Thus, antagonists of thepresent invention could serve as a valuable therapeutic to reduceinflammation and pathological effects in IBD and related diseases.

Crohn's disease is a chronic, relapsing-remitting inflammatory diseaseof the intestinal tract. It is a chronic disease that often occurs at anearly age, thus requiring patients to be treated for decades. Incontrast to the more limited tissue layer involvement of ulcerativecolitis (i.e. mucosa and submucosa of the colon), Crohn's disease canextend through all layers of the intestinal wall of both the large andsmall intestine. Symptoms of Crohn's disease include diarrhea, weightloss, blood, and abdominal pain: Complications are very serious andinclude intestinal fistulas, abscesses, and obstructions.

In healthy people, there is continuous, clinically undetected immuneresponse to these antigens. In Crohn's disease, the response isprolonged and amplified. The precise cause nor the specific antigenictrigger has been identified, though it has been hypothesized to be acombination of a genetic predisposition plus an environmental trigger,such as exposure to endogenous or exogenous intestinal antigens.Therefore, in an effort to inhibit this characteristic aberrant immuneresponse, immunosuppressive agents are used to treat CD patients.However, it is clear from the side effects of these drugs and theresponse/failure rate, that more selective and efficacious therapies areneeded.

Many immune cells, including neutrophils, macrophages, B and Tlymphocytes, and mast cell, are present in the mucosal layer of healthyintestines. The intact epithelium lining the mucosa prevents these cellsfrom being overstimulated by the large antigenic load to which the GItract is exposed on a daily basis. It is thought that Crohn's diseasepatients have increased intestinal permeability (perhaps because of theabove mentioned genetic predisposition) that exposes the immune cells tonumerous antigens. IL-23 and IL-17 appear to play a role in theoveractive response and ensuing disease progression.

Ulcerative colitis (UC) is an inflammatory disease of the largeintestine, commonly called the colon, characterized by inflammation andulceration of the mucosa or innermost lining of the colon. Thisinflammation causes the colon to empty frequently, resulting indiarrhea. Symptoms include loosening of the stool and associatedabdominal cramping, fever and weight loss.

Although the exact cause of UC is unknown, recent research suggests thatthe body's natural defenses are operating against proteins in the bodywhich the body thinks are foreign (an “autoimmune reaction”). Perhapsbecause they resemble bacterial proteins in the gut, these proteins mayeither instigate or stimulate the inflammatory process that begins todestroy the lining of the colon. As the lining of the colon isdestroyed, ulcers form releasing mucus, pus and blood. The diseaseusually begins in the rectal area and may eventually extend through theentire large bowel. Repeated episodes of inflammation lead to thickeningof the wall of the intestine and rectum with scar tissue. Death of colontissue or sepsis may occur with severe disease. The symptoms ofulcerative colitis vary in severity and their onset may be gradual orsudden. Attacks may be provoked by many factors, including respiratoryinfections or stress.

Although there is currently no cure for UC available, treatments arefocused on suppressing the abnormal inflammatory process in the colonlining. Treatments including corticosteroids, immunosuppressives (eg.azathioprine, mercaptopurine, and methotrexate) and aminosalicytates areavailable to treat the disease. However, the long-term use ofimmunosuppressives such as corticosteroids and azathioprine can resultin serious side effects including thinning of bones, cataracts,infection, and liver and bone marrow effects. Ti the patients in whomcurrent therapies are not successful, surgery is an option. The surgeryinvolves the removal of the entire colon and the rectum.

There are several animal models that can partially mimic chroniculcerative colitis. Some of the most widely used models are the is the2,4,6-trinitrobenesulfonic acid/ethanol (TNBS) induced colitis model orthe oxazalone model, which induce chronic inflammation and ulceration inthe colon. When TNBS or oxazalone is introduced into the colon ofsusceptible mice via intra-rectal instillation, it induces T-cellmediated immune response in the colonic mucosa, in this case leading toa massive mucosal inflammation characterized by the dense infiltrationof T-cells and macrophages throughout the entire wall of the largebowel. Moreover, this histopathologic picture is accompanies by theclinical picture of progressive weight loss (wasting), bloody diarrhea,rectal prolapse, and large bowel wall thickening (Neurath et al. Intern.Rev. Immunol. 19:51-62, 2000).

Another colitis model uses dextran sulfate sodium (DSS), which inducesan acute colitis manifested by bloody diarrhea, weight loss, shorteningof the colon and mucosal ulceration with neutrophil infiltration.DSS-induced colitis is characterized histologically by infiltration ofinflammatory cells into the lamina propria, with lymphoid hyperplasia,focal crypt damage, and epithelial ulceration. These changes are thoughtto develop due to a toxic effect of DSS on the epithelium and byphagocytosis of lamina propria cells and production of TNF-alpha andIFN-gamma. Despite its common use, several issues regarding themechanisms of DSS about the relevance to the human disease remainunresolved. DSS is regarded as a T cell-independent model because it isobserved in T cell-deficient animals such as SCID mice.

The administration of antagonists of the present invention to theseTNBS, DSS, oxazalone, or T cell transfer models can be used to evaluatethe use of those antagonists to ameliorate symptoms and alter the courseof gastrointestinal disease. Moreover, the results showing inhibition ofIL-17 and IL-23 signalling provide proof of concept that otherIL-17/IL-23 antagonists can also be used to ameliorate symptoms in thecolitis/IBD models and alter the course of disease. See Example 28.

3. Psoriasis

Psoriasis is a chronic skin condition that affects more than sevenmillion Americans. Psoriasis occurs when new skin cells grow abnormally,resulting in inflamed, swollen, and scaly patches of skin where the oldskin has not shed quickly enough. Plaque psoriasis, the most commonform, is characterized by inflamed patches of skin (“lesions”) toppedwith silvery white scales. Psoriasis may be limited to a few plaques orinvolve moderate to extensive areas of skin, appearing most commonly onthe scalp, knees, elbows and trunk. Although it is highly visible,psoriasis is not a contagious disease. The pathogenesis of the diseasesinvolves chronic inflammation of the affected tissues. IL-17 and IL-23are both overexpressed in psoriatic skin compared to non-psoriatic skin(Li et al, J Huazhong Univ Sci Technolog Med Sci 24:294 (2004); Piskinet al, J Immunol. 176:1908 (2006)). Therefore, antagonists of thepresent invention could serve as a valuable therapeutic to reduceinflammation and pathological effects in psoriasis, other inflammatoryskin diseases, skin and mucosal allergies, and related diseases.

Psoriasis is a T-cell mediated inflammatory disorder of the skin thatcan cause considerable discomfort. It is a disease for which there is nocure and affects people of all ages. Psoriasis affects approximately twopercent of the populations of European and North America. Althoughindividuals with mild psoriasis can often control their disease withtopical agents, more than one million patients worldwide requireultraviolet or systemic immunosuppressive therapy. Unfortunately, theinconvenience and risks of ultraviolet radiation and the toxicities ofmany therapies limit their long-term use. Moreover, patients usuallyhave recurrence of psoriasis, and in some cases rebound, shortly afterstopping immunosuppressive therapy.

In addition to other disease models described herein, the activity ofantagonists of the present invention on inflammatory tissue derived fromhuman psoriatic lesions can be measured in vivo using a severe combinedimmune deficient (SCID) mouse model. Several mouse models have beendeveloped in which human cells are implanted into immunodeficient mice(collectively referred to as xenograft models); see, for example, CaftanA R, Douglas E, Leuk. Res. 18:513-22 (1994) and Flavell, D J,Hematological Oncology 14:67-82 (1996). As an in vivo xenograft modelfor psoriasis, human psoriatic skin tissue is implanted into the SODmouse model, and challenged with an appropriate antagonist. Moreover,other psoriasis animal models in the art may be used to evaluate thepresent antagonists, such as human psoriatic skin grafts implanted intoAGR129 mouse model, and challenged with an appropriate antagonist (e.g.,see, Boyman, O. et al., J. Exp. Med. 199:731 (2004), incorporated hereinby reference). IL-17/IL-23 antibodies or binding peptides that bind,block, inhibit, reduce, antagonize or neutralize the activity of IL-17,IL-23 or both IL-17 and IL-23 are preferred antagonists. Similarly,tissues or cells derived from human colitis, IBD, arthritis, or otherinflammatory lesions can be used in the SCID model to assess theanti-inflammatory properties of the IL-17 and IL-23 antagonistsdescribed herein.

Efficacy of treatment is measured and statistically evaluated asincreased anti-inflammatory effect within the treated population overtime using methods well known in the art. Some exemplary methodsinclude, but are not limited to measuring for example, in a psoriasismodel, epidermal thickness, the number of inflammatory cells in theupper dermis, and the grades of parakeratosis. Such methods are known inthe art and described herein. For example, see Zeigler, M. et al. LabInvest 81:1253 (2001); Zollner, T. M. et al. J. Clin. Invest. 109:671(2002); Yamanaka, N. et al. Microbiol. Immunol. 45:507 (2001);Raychaudhuri, S. P. et al. Br. J. Dermatol. 144:931 (2001); Boehncke, W.H et al. Arch. Dermatol. Res. 291:104, (1999); Boehncke, W. H et al. J.Invest. Dermatol. 116:596 (2001); Nickoloff, B. J. et al. Am. Pathol.146:580 (1995); Boehncke, W. H et al. J. Cutan. Pathol. 24:1, (1997);Sugai, J., M. et al. Dermatol. Sci. 17:85 (1998); and Villadsen L. S. etal. J. Clin. Invest. 112:1571 (2003). Inflammation may also be monitoredover time using well-known methods such as flow cytometry (or PCR) toquantitate the number of inflammatory or lesional cells present in asample, score (weight loss, diarrhea, rectal bleeding, colon length) forIBD. For example, therapeutic strategies appropriate for testing in sucha model include direct treatment using IL-17 and IL-23 antagonists(singly or together), or related conjugates or antagonists based on thedisrupting interaction of IL-17 and IL-23 with their receptors.

4. Atopic Dermatitis.

AD is a common chronic inflammatory disease that is characterized byhyperactivated cytokines of the helper T cell subset 2 (Th2). Althoughthe exact etiology of AD is unknown, multiple factors have beenimplicated, including hyperactive Th2 immune responses, autoimmunity,infection, allergens, and genetic predisposition. Key features of thedisease include xerosis (dryness of the skin), pruritus (itchiness ofthe skin), conjunctivitis, inflammatory skin lesions, Staphylococcusaureus infection, elevated blood eosinophilia, elevation of serum IgEand IgG1, and chronic dermatitis with T cell, mast cell, macrophage andeosinophil infiltration. Colonization or infection with S. aureus hasbeen recognized to exacerbate AD and perpetuate chronicity of this skindisease.

AD is often found in patients with asthma and allergic rhinitis, and isfrequently the initial manifestation of allergic disease. About 20% ofthe population in Western countries suffer from these allergic diseases,and the incidence of AD in developed countries is rising for unknownreasons. AD typically begins in childhood and can often persist throughadolescence into adulthood. Current treatments for AD include topicalcorticosteroids, oral cyclosporin A, non-corticosteroidimmunosuppressants such as tacrolimus (FK506 in ointment form), andinterferon-gamma. Despite the variety of treatments for AD, manypatients' symptoms do not improve, or they have adverse reactions tomedications, requiring the search for other, more effective therapeuticagents. The antagonists of the present invention can be used toneutralize IL-17 and IL-23 (via p19) in the treatment of specific humandiseases such as atoptic dermatitis, inflammatory skin conditions, andother inflammatory conditions disclosed herein.

5. Asthma

IL-17 plays an important role in allergen-induced T cell activation andneutrophilic influx in the airways. The receptor for IL-17 is expressedin the airways (Yao, et al. Immunity 3:811 (1995)) and IL-17 mediatedneutrophil recruitment in allergic asthma is largely induced by thechemoattractant IL-8, GRO-alpha and macrophage inflammatory protein-2(MIP-2) produced by IL-17 stimulated human bronchial epithelial cells(HBECs) and human bronichial fibroblasts (Yao, et al. J Immunol 155:5483(1995)); Molet, et al. J Allergy Clin Immunol 108:430 (2001)). IL-17also stimulates HBECs to release IL-6, a neutrophil-activating factor(Fossiez, et al, J Exp Med 183:2593 (1996), and Linden, et al. Int ArchAllergy Immunol 126:179 (2001)) and has been shown to synergize withTNF-alpha to prolong the survival of human neutrophils in vitro (Lana,et al. Eur Respir J 21:387 (2003)). Moreover, IL-17 is capable ofamplifying the inflammatory responses in asthma by its ability toenhance the secretion of cytokines implicated in airway remodeling suchas the profibrotic cytokines, IL-6 and IL-11 and inflammatory mediatorsgranulocyte colony-stimulating factor (G-CSF) and granulocyte macrophagecolony-stimulating factor (GM-CSF) (Molet, et al. J Allergy Clin Immunol108:430 (2001)).

Clinical evidence shows that acute, severe exacerbations of asthma areassociated with recruitment and activation of neutrophils in theairways, thus IL-17 is likely to play a significant role in asthma.Furthermore, since IL-23 is important in the maintenance anddifferentiation of IL-1.7 producing cells (e.g. Th17 cells), IL-23 isalso likely to play a role in asthma. Patients with mild asthma displaya detectable increase in the local concentration of free, soluble IL-17protein (Molet, et al. J Allergy Clin Immunol 108:430 (2001)) whilehealthy human volunteers with induced, severe airway inflammation due tothe exposure to a swine confinement, display a pronounced increase inthe concentration of free, soluble IL-17 protein in the bronchoalveolarspace (Fossiez et al, J Exp Med 183:2593 (1996), and Linden, et al. IntArch Allergy Immunol 126:179 (2001)). Furthermore, IL-17 levels insputum have correlated with individuals who have increased airwayhyper-reactivity Barezyk, et al. Respir Med 97:726 (2003).

In animal models of airway hyper-responsiveness, chronic inhalation ofovalbumin by sensitized mice resulted in bronchial eosinophilicinflammation and early induction of IL-17 mRNA expression in inflamedlung tissue, together with a bronchial neutrophilia Hellings, et al. AmJ Respir Cell Mol Biol 28:42 (2003). Anti-IL-17 monoclonal antibodiesstrongly reduced bronchial neutrophilic influx but significantlyenhanced IL-5 levels in both bronchoalveolar lavage fluid and serum, andaggravated allergen-induced bronchial eosinophilic influx, suggestingthat IL-17 may be involved in determining the balance between neutrophiland eosinophil accumulation following antigen insult Id.

Apart from asthma, several chronic inflammatory airway diseases arecharacterized by neutrophil recruitment in the airways and both IL-17and IL-23 have been reported to play an important role in thepathogenesis of respiratory conditions such as chronic obstructivepulmonary disease (COPD), bacterial pneumonia and cystic fibrosis(Linden, et al. Eur Respir J 15:973 (2000), Ye, et al. Am J Respir CellMol Biol 25:335 (2001), Rahman, et al. Clin Immunol 115:268 (2005);Dubin and Kolls, Am. J. Physiol. Lung Cell Mol. Physiol. 292: L519-28(2007); McAllister et al. J. Immunol. 175:404-412 (2005)). An anti-IL-17and/or anti-IL-23 therapeutic molecule could be demonstrated to beefficacious for chronic inflammatory airway disease in an in vitro modelof inflammation. The ability of antagonists to IL-17 and/or IL-23activity to inhibit IL-17 or and/or IL-23 signalling to induce cytokineand chemokine production from cultured HBECs or bronchial fibroblastscould be used as a measure of efficacy for such antagonists in theprevention of the production of inflammatory mediators directlyresulting from IL-17 and/or IL-23 stimulation. If the addition ofantagonists to IL-17 and/or IL-23 activity markedly reduces theproduction and expression of inflammatory mediators, it would beexpected to be efficacious in inflammatory aspects associated withchronic airway inflammation.

6. Multiple Sclerosis

Multiple sclerosis is a relatively commonly occurring autoimmune diseasecharacterized by demyelination and chronic inflammation of the centralnervous system (CNS). Although the mechanisms underlying diseaseinitiation are not clearly understood, the disease processes thatcontribute to clinical progression of multiple sclerosis areinflammation, demyelination, and axonal loss, or neurodegeneration.Macrophages and microglia are the main immune cells of the CNS. Thesecells, as well as T cells, neutrophils, astrocytes, and microglia, cancontribute to the immune-related pathology of, e.g., multiple sclerosis.Furthermore, T cell reactivity/autoimmunity to several myelin proteins,including myelin basic protein (MBP), proteolipid protein (PLP), myelinoligodendrocyte protein (MOO), and perhaps other myelin proteins, havebeen implicated in the induction and perpetuation of disease state andpathology of multiple sclerosis. This interaction of autoreactive Tcells and myelin proteins can result in the release of proinflammatorycytokines, including TNT-a, IFN-g, and IL-17, among others. Additionalconsequences are the proliferation of T cells, activation of B cells andmacrophages, upregulation of chemokines and adhesion molecules, and thedisruption of the blood-brain barrier. The ensuing pathology is a lossof oligodendrocytes and axons, and the formation of a demyelinated“plaque”. The plaque consists of a lesion in which the myelin sheath isnow absent and the demyelinated axons are embedded within glial scartissue. Demyelination can also occur as the result of specificrecognition and opsinization of myelin antigens by autoantibodies,followed by complement- and/or activated macrophage-mediateddestruction. It is this axonal loss and neurodegeneration that isthought to be primarily responsible for the irreversible neurologicalimpairment that is observed in progressive multiple sclerosis.

There is a large amount of clinical and pathological heterogeneity inthe course of human multiple sclerosis. Symptoms most often beginbetween the ages of 18 and 50 years old, but can begin at any age. Theclinical symptoms of multiple sclerosis can vary from mild visiondisturbances and headaches, to blindness, severe ataxia and paralysis.The majority of the patients (approximately 70-75%) haverelapsing-remitting multiple sclerosis, in which disease symptoms canrecur within a matter of hours to days, followed by a much slowerrecovery; the absence of symptoms during stages of remission is notuncommon. The incidence and frequency of relapses and remissions canvary greatly, but as time progresses, the recovery phases can beincomplete and slow to occur. This worsening of disease in these casesis classified as secondary-progressive multiple sclerosis, and occurs inapproximately 10-15% of multiple sclerosis patients. Another 10-15% ofpatients are diagnosed with primary-progressive multiple sclerosis, inwhich disease symptoms and physical impairment progress at a steady ratethroughout the disease process.

Both IL-23 and IL-17 are overexpressed in the central nervous system ofhumans with multiple sclerosis and in mice undergoing an animal model ofmultiple sclerosis, experimental autoimmune encephalomyelitis (EAE).(See Example 11). The overexpression is observed in mice when the EAE isinduced by either myelin oligodendrocyte glycoprotein (MOG) 35-55peptide- or proteolipid peptide (PLP). Furthermore, neutralization ofeither IL-23/p19 or IL-17 results in amelioration of EAE symptoms inmice (Park et al, Nat Immunol. 6:1133 (2005); Chen et al, J Clin Invest.116:1317 (2006)).

The ability of antagonists to IL-17 and/or IL-23 activity to inhibitIL-17 or and/or IL-23 signalling-induced cytokine and chemokineproduction could be used as a measure of efficacy for such antagonistsin the treatment of multiple sclerosis. The addition of antagonists toIL-17 and/or IL-23 activity markedly reduces the production andexpression of inflammatory mediators (i.e. CNS-infiltrating immunecells; CNS expression of inflammatory cytokines/chemokines, etc.) andsymptoms of multiple sclerosis (e.g. paralysis; ataxia; weight loss,etc). See Example 8. These results indicate that antagonists to IL-17and/or IL-23 activity would be efficacious in the treatment of humans.

7. Cancer

Chronic inflammation has long been associated with increased incidenceof malignancy and similarities in the regulatory mechanisms have beensuggested for more than a century. Infiltration of innate immune cells,elevated activities of matrix metalloproteases (MMP) and increasedangiogenesis and vasculature density are a few examples of thesimilarities between chronic and tumour-associated inflammation.Conversely, the elimination of early malignant lesions by immunesurveillance, which relies on the cytotoxic activity oftumour-infiltrating T cells or intra-epithelial lymphocytes, is thoughtto be rate-limiting for the risk to develop cancer.

There are numerous publications describing important roles for IL-23 andIL-17 in tumor biology and/or angiogenesis. Both IL-23 and IL-17 havebeen published to be upregulated in several human tumors and cancers,including but not limited to those of the colon, breast, ovarian,cervical, prostate, lung, and stomach, as well as melanoma and T celllymphoma (Tartour et al, Cancer Res. 59:3698 (1999); Kato et al,Biochem. Biophys. Res. Commun. 282:735 (2001); Steiner et al, Prostate.56:171 (2003); Langowksi et al, Nature, 442:461-5, (2006)). Thus,neutralization of both IL-17 and a key upstream regulator of IL-17,IL-23 (via p19), is a potent and effective means of treating cancer andother neoplastic diseases. Therefore, neutralizing both IL-17 and IL-23with antagonists of the present invention (i.e. a single neutralizingentity or antibody to IL-17 and IL-23 or an antagonistic molecule thatwill neutralize both together, such as a bispecific antibody orbispecific scFv) will have better efficacy in these diseases thanantagonists directed toward either of IL-17 or IL-23 alone.

Angiogenesis refers to the formation of new capillaries from preexistingvessels. There are several reports that angiogenesis plays importantroles in hematological malignancies and solid tumors. The initiation ofangiogenesis and the switch to the angiogenic phenotype requires achange between proangiogenic factors and angiogenic inhibitors (Folkman,Nat. Med. 1:27 (1995)). IL-17 acts as a stimulatory hematopoieticcytokine by initiating proliferation of mature neutrophils and byexpanding myeloid progenitors. It has been well documented that IL-17has pro-angiogenic activities and stimulates the migration of vascularendothelial cells, which are associated with tumor promotion (Numasakiet al, Blood, 101:2620 (2003); Yang et al, J. Biol. Chem., 278:33232(2003); Fujino et al, Gut, 52:65 (2003)). In vitro angiogenic activitycan be suppressed by neutralizing IL-17 with a neutralizing anti-IL-17monoclonal antibody, further supporting the role of IL-17 in thisaction. It is also able to selectively enhance mitogenic activity ofbasic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF),and vascular endothelial growth factor (VEGF), and IL-17 may alsopromote bFGF-, HGF- and VEGF-mediated angiogenesis via bFGF-, HGF- andVEGF-induced growth of vascular endothelial cells (Takahashi et al,Immunol Lett. 98:189 (2005)). IL-17 has been reported to augment thesecretion of several angiogenic CXC chemokines (e.g. CXCL1, CXCL5,CXCL6, and CXCL8) in non-small cell lung cancer (NSCLC) lines.Endothelial cell chemotactic activity (a measure of net angiogenicpotential) is increased in response to conditioned medium from NSCLCstimulated with recombinant IL-17. NSCLC lines transfected with IL-17grew more rapidly versus controls when transplanted in SCID mice(Numasaki et al, J Immunol. 175:6177 (2005)). Furthermore, IL-17 hasbeen reported to be associated with increased IL-6 at the site of tumorsand is well reported to increase MMP-9 expression. MMP-9 is an importantmodulator in diseases of inflammation, autoimmunity, and cancer. Thesereports, therefore, clearly implicate a pro-angiogenic and tumorpromoting action for IL-17. Therefore, neutralizing both IL-17 and IL-23with antagonists of the present invention (i.e. a singleneutralizing-entity or antibody to IL-17 and IL-23 or an antagonisticmolecule that will neutralize both together, such as a bispecificantibody or bispecific scFv) will have better efficacy than antagonistsdirected toward either of IL-17 or IL-23 alone.

Similar to IL-17, IL-23 promotes inflammatory responses includingupregulation of MMP-9, and is also reported to increase angiogenesis andreduce CD8+ T-cell infiltration. Taken together, these actions can leadto enhanced initiation, progression, and/or maintenance of tumors,cancers, and other transformed growths. That IL-23 plays an importantrole in cancerous diseases is supported by the observation thatneutralization of IL-23 with a monoclonal antibody or with geneticdeletion in mice reduces tumor growth in several murine tumor models(Langowksi et al. Nature, 442: 461-5 (2006)). Efficacy is associatedwith reduced IL-17 expression and reductions in IL-17-relatedtumorigenic biomarkers, such as granulocyte infiltration, G-CSF andMMP-9. Therefore, neutralizing both IL-17 and IL-23 with antagonists ofthe present invention (i.e. a single neutralizing entity or antibody toIL-17 and IL-23 or an antagonistic molecule that will neutralize bothtogether, such as a bispecific antibody or bispecific scFv) will havebetter efficacy in these diseases than antagonists directed towardeither of IL-17 or IL-23 alone.

8. Irritable Bowel Syndrome (IBS)

Irritable bowel syndrome (IBS) represents a disease characterized byabdominal pain or discomfort and an erratic bowel habit. IBS patientscan be characterized into three main groups based on bowel habits: thosewith predominantly loose or frequent stools, those with predominantlyhard or infrequent stools, and those with variable or normal stools(Talley et al., Expert Opin. Emerg. Drugs 7:91-8 (2002)). Alteredintestinal motility, abnormalities in epithelial function, abnormaltransit of stool and gas, and stress, may contribute to symptoms, whilevisceral hypersensitivity is a key feature in most patients. Geneticfactors affecting pain-signaling and disturbances in central processingof afferent signals are postulated to predispose individuals to IBSfollowing specific environmental exposures. Studies have alsodemonstrated that inflammatory responses in the colon may contribute toincreased sensitivity of smooth muscle and enteric nerves and thereforeperturb sensory-motor functions in the intestine (Collins et al., Can.J. Gastroenterol. 15 Suppl. B:14B-16B (2001)). There is clinical overlapbetween IBS and IBD, with IBS-like symptoms frequently reported inpatients before the diagnosis of IBD, and a higher than expected IBSsymptoms in patients in remission from established IBD. Thus, theseconditions may coexist with a higher than expected frequency, or mayexist on a continuum, with IBS and IBD at different ends of the samespectrum. However, it should be noted that in most IBS patients, colonicbiopsy specimens appear normal. Nevertheless, IBS significantly affectsa very large number of individuals (U.S. prevalence in 2000,approximately 16 million individuals), resulting in a total cost burdenof 1.7 billion dollars (year 2000). Thus, among the most prevalent andcostly gastrointestinal diseases and disorders, IBS is second only togastroesophageal reflux disease (GERD). Yet unlike GERD, treatment forIBS remains unsatisfactory ((Talley et al., Expert Opin. Emerg. Drugs7:91-8 (2002)); Farhadi et al., Expert Opin. Investig. Drugs 10:1211-22(2001); Collins et al., Can. J. Gastroenterol. 15 Suppl. B:14B-16B(2001)), demonstrating that IBS clearly represents an unmet medicalneed.

Converging disease models have been proposed that postulate an enhancedresponsiveness of neural, immune or neuroimmune circuits in the centralnervous system (CNS) or in the gut to central (psychosocial) orperipheral (tissue irritation, inflammation, infection) perturbations ofnormal homeostasis (Talley et al., Expert. Opin. Emerg. Drugs 7:91-8(2002)). This enhanced responsiveness results in dysregulation of gutmotility, epithelial function (immune, permeability), and visceralhypersensitivity, which in turn results in IBS symptoms.

There may be a role for a number of different molecules in thepathogenesis of IBS including a role for molecules that stimulateneurons and those that are involved in initiation of inflammatoryprocess, including IL-17A and IL-23p19.

Efficacy of inhibitors of these molecules could be tested in vivo inanimal models of disease. Several animal models have been proposed thatmimic key features of IBS and involve centrally targeted stimuli(stress) or peripherally targeted stimuli (infection, inflammation). Twoexamples of in vivo animal models that can be used to determine theeffectiveness of inhibitors in the treatment of IBS are (i) modelsfocusing on primary CNS-directed pathogeneisis of IBS (stress models),and (ii) models focusing on gut-directed inducers of stress (i.e. gutinflammation, infection or physical stress). It should be noted however,that events within the CNS or in the gastrointestinal (GI) tract do notoccur in isolation and that symptoms of IBS most likely result from acomplex interaction between signals from the CNS On the GI and viceversa.

Thus, in summary, there are several molecules and pathogenic pathwaysthat are shared by IL-17 and IL-23 which play important roles in thedevelopment, progression, and maintenance of both autoimmune diseasesand cancerous diseases. These include the pro-angiogenic roles of IL-17and IL-23; enhanced MMP-9 levels and activity by IL-17 and IL-23; IL-23,TGF-b and IL-6-mediated production and/or maintenance of Th17 cells;roles of TGF-b and IL-6 in the generation of Foxp3+ regulatory T cells;and additional pathways and molecules. Therefore, the IL-17/IL-23 axisrepresents an important link to the inappropriate and pathogenic T cellresponses associated with autoimmune diseases, tumour-promotingpro-inflammatory processes, and the failure of the adaptive immunesurveillance to infiltrate tumours. Therefore, neutralizing both IL-17and IL-23 with antagonists of the present invention (i.e. a singleneutralizing entity or antibody to IL-17 and IL-23 or an antagonisticmolecule that will neutralize both together, such as a bispecificantibody or bispecific scFv) will have better efficacy in these diseasesthan antagonists directed toward either of IL-17 or IL-23 alone.

J) Pharmaceutical Compositions

For pharmaceutical use, the antibodies of the present invention areformulated for parenteral, particularly intravenous or subcutaneous,delivery according to conventional methods. Intravenous administrationwill be by bolus injection, controlled release, e.g, using mini-pumps orother appropriate technology, or by infusion over a typical period ofone to several hours. In general, pharmaceutical formulations willinclude a hematopoietic protein in combination with a pharmaceuticallyacceptable vehicle, such as saline, buffered saline, 5% dextrose inwater or the like. Formulations may further include one or moreexcipients, preservatives, solubilizers, buffering agents, albumin toprevent protein loss on vial surfaces, etc. When utilizing such acombination therapy, the cytokines may be combined in a singleformulation or may be administered in separate formulations. Methods offormulation are well known in the art and are disclosed, for example, inRemington's Pharmaceutical Sciences, Gennaro, ed., Mack Publishing Co.,Easton Pa., 1990, which is incorporated herein by reference. Therapeuticdoses will generally be in the range of 0.1 to 100 mg/kg of patientweight per day, preferably 0.5-20 mg/kg per day, with the exact dosedetermined by the clinician according to accepted standards, taking intoaccount the nature and severity of the condition to be treated, patienttraits, etc. Determination of dose is within the level of ordinary skillin the art. The proteins will commonly be administered over a period ofup to 28 days following chemotherapy or bone-marrow transplant or untila platelet count of >20,000/mm³, preferably >50,000/mm³, is achieved.More commonly, the proteins will be administered over one week or less,often over a period of one to three days. In general, a therapeuticallyeffective amount of antibodies of the present invention is an amountsufficient to produce a clinically significant increase in theproliferation and/or differentiation of lymphoid or myeloid progenitorcells, which will be manifested as an increase in circulating levels ofmature cells (e.g. platelets or neutrophils). Treatment of plateletdisorders will thus be continued until a platelet count of at least20,000/mm³, preferably 50,000/mm³, is reached. The antibodies of thepresent invention can also be administered in combination with othercytokines such as IL-3, -6 and -11; stem cell factor; erythropoietin;G-CSF and GM-CSF. Within regimens of combination therapy, daily doses ofother cytokines are commonly known by one skilled in the art, or can bedetermined without undue experimentation. Combination therapy with EPO,for example, is indicated in anemic patients with low EPO levels.

Generally, the dosage of administered antibodies will vary dependingupon such factors as the patient's age, weight, height, sex, generalmedical condition and previous medical history. Typically, it isdesirable to provide the recipient with a dosage of antibodies which isin the range of from about 1 pg/kg to 10 mg/kg (amount of agent/bodyweight of patient), although a lower or higher dosage also may beadministered as circumstances dictate.

Administration of antibodies of the invention to a subject can beintravenous, intraarterial, intraperitoneal, intramuscular,subcutaneous, intrapleural, intrathecal, by perfusion through a regionalcatheter, or by direct intralesional injection. When administeringtherapeutic proteins by injection, the administration may be bycontinuous infusion or by single or multiple boluses.

Additional routes of administration include oral, mucosal-membrane,pulmonary, and transcutaneous. Oral delivery is suitable for polyestermicrospheres, zein microspheres, proteinoid microspheres,polycyanoacrylate microspheres, and lipid-based systems (see, forexample, DiBase and Morrel, “Oral Delivery of MicroencapsulatedProteins,” in Protein Delivery: Physical Systems, Sanders and Hendren(eds.), pages 255-288 (Plenum Press 1997)). The feasibility of anintranasal delivery is exemplified by such a mode of insulinadministration (see, for example, Hinchcliffe and Ilium, Adv. DrugDeliv. Rev. 35:199 (1999)). Dry or liquid particles comprisingantibodies of the invention can be prepared and inhaled with the aid ofdry-powder dispersers, liquid aerosol generators, or nebulizers (e.g.,Pettit and Gombotz, TIBTECH 16:343 (1998); Patton et al., Adv. DrugDeliv. Rev. 35:235 (1999)). This approach is illustrated by the AERXdiabetes management system, which is a hand-held electronic inhaler thatdelivers aerosolized insulin into the lungs. Studies have shown thatproteins as large as 48,000 kDa have been delivered across skin attherapeutic concentrations with the aid of low-frequency ultrasound,which illustrates the feasibility of transcutaneous administration(Mitragotri et al., Science 269:850 (1995)). Transdermal delivery usingelectroporation provides another means to administer a molecule havingIL-17 and IL-23/p19 binding activity (Potts et al., Pharm. Biotechnol.10:213 (1997)).

A pharmaceutical composition comprising one or more antibodies of theinvention can be formulated according to known methods to preparepharmaceutically useful compositions, whereby the therapeutic proteinsare combined in a mixture with a pharmaceutically acceptable carrier. Acomposition is said to be a “pharmaceutically acceptable carrier” if itsadministration can be tolerated by a recipient patient. Sterilephosphate-buffered saline is one example of a pharmaceuticallyacceptable carrier. Other suitable carriers are well-known to those inthe art. See, for example, Gennaro (ed.), Remington's PharmaceuticalSciences, 19th Edition (Mack Publishing Company 1995).

For purposes of therapy, antibodies of the invention and apharmaceutically acceptable carrier are administered to a patient in atherapeutically effective amount. A combination of a therapeuticmolecule of the present invention and a pharmaceutically acceptablecarrier is said to be administered in a “therapeutically effectiveamount” if the amount administered is physiologically significant. Anagent is physiologically significant if its presence results in adetectable change in the physiology of a recipient patient. For example,an agent used to treat inflammation is physiologically significant ifits presence alleviates the inflammatory response. Effective treatmentmay be assessed in a variety of ways. In one embodiment, effectivetreatment is determined by reduced inflammation. In other embodiments,effective treatment is marked by inhibition of inflammation. In stillother embodiments, effective therapy is measured by increased well-beingof the patient including such signs as weight gain, regained strength,decreased pain, thriving, and subjective indications from the patient ofbetter health.

A pharmaceutical composition comprising antibodies of the invention canbe furnished in liquid form, in an aerosol, or in solid form. Liquidforms, are illustrated by injectable solutions and oral suspensions.Exemplary solid forms include capsules, tablets, and controlled-releaseforms. The latter form is illustrated by miniosmotic pumps and implants(Bremer et al., Pharm. Biotechnol. 10:239 (1997); Ranade, “Implants inDrug Delivery,” in Drug Delivery Systems, Ranade and Hollinger (eds.),pages 95-123 (CRC Press 1995); Bremer et al., “Protein Delivery withInfusion Pumps,” in Protein. Delivery: Physical Systems, Sanders andHendren (eds.), pages 239-254 (Plenum Press 1997); Yewey et al.,“Delivery of Proteins from a Controlled Release Injectable Implant,” inProtein Delivery: Physical Systems, Sanders and Hendren (eds.), pages93-117 (Plenum Press 1997)).

Liposomes provide one means to deliver therapeutic polypeptides to asubject intravenously, intraperitoneally, intrathecally,intramuscularly, subcutaneously, or via oral administration, inhalation,or intranasal administration. Liposomes are microscopic vesicles thatconsist of one or more lipid bilayers surrounding aqueous compartments(see, generally, Bakker-Woudenberg et al., Eur. J. Clin. Microbial.Infect. Dis. 12 (Suppl. 1):S61 (1993), Kim, Drugs 46:618 (1993), andRanade, “Site-Specific Drug Delivery Using Liposomes as Carriers,” inDrug Delivery Systems, Ranade and Hollinger (eds.), pages 3-24 (CRCPress 1995)). Liposomes are similar in composition to cellular membranesand as a result, liposomes can be administered safely and arebiodegradable. Depending on the method of preparation, liposomes may beunilamellar or multilamellar, and liposomes can vary in size withdiameters ranging from 0.02 μm to greater than 10 μM. A variety ofagents can be encapsulated in liposomes: hydrophobic agents partition inthe bilayers and hydrophilic agents partition within the inner aqueousspace(s) (see, for example, Machy et al., Liposomes In Cell Biology AndPharmacology (John Libbey 1987), and Ostro et al., American J. Hosp.Pharm. 46:1576 (1989)). Moreover, it is possible to control thetherapeutic availability of the encapsulated agent by varying liposomesize, the number of bilayers, lipid composition, as well as the chargeand surface characteristics of the liposomes.

Liposomes can adsorb to virtually any type of cell and then slowlyrelease the encapsulated agent. Alternatively, an absorbed liposome maybe endocytosed by cells that are phagocytic. Endocytosis is followed byintralysosomal degradation of liposomal lipids and release of theencapsulated agents (Scherphof et al., Ann. N.Y. Acad. Sci. 446:368(1985)). After intravenous administration, small liposomes (0.1 to 1.0μm) are typically taken up by cells of the reticuloendothelial system,located principally in the liver and spleen, whereas liposomes largerthan 3.0 μm are deposited in the lung. This preferential uptake ofsmaller liposomes by the cells of the reticuloendothelial system hasbeen used to deliver chemotherapeutic agents to macrophages and totumors of the liver.

The reticuloendothelial system can be circumvented by several methodsincluding saturation with large doses of liposome particles, orselective macrophage inactivation by pharmacological means (Claassen etal., Biochim. Biophys. Acta 802:428 (1984)). In addition, incorporationof glycolipid- or polyethelene glycol-derivatized phospholipids intoliposome membranes has been shown to result in a significantly reduceduptake by the reticuloendothelial system (Allen et al., Biochim.Biophys. Acta 1068:133 (1991); Allen et al., Biochim. Biophys, Acta1150:9 (1993)).

Liposomes can also be prepared to target particular cells or organs byvarying phospholipid composition or by inserting receptors or ligandsinto the liposomes. For example, liposomes, prepared with a high contentof a nonionic surfactant, have been used to target the liver (Hayakawaet al., Japanese Patent 04-244,018; Kato et al., Biol. Pharm. Bull.16:960 (1993)). These formulations were prepared by mixing soybeanphosphatidylcholine, α-tocopherol, and ethoxylated hydrogenated castoroil (HCO-60) in methanol, concentrating the mixture under vacuum, andthen reconstituting the mixture with water. A liposomal formulation ofdipalmitoylphosphatidylcholine (DPPC) with a soybean-derivedsterylglucoside mixture (SG) and cholesterol (Ch) has also been shown totarget the liver (Shimizu et al., Biol. Pharm. Bull. 20:881 (1997)).

Alternatively, various targeting ligands can be bound to the surface ofthe liposome, such as antibodies, antibody fragments, carbohydrates,vitamins, and transport proteins. For example, liposomes can be modifiedwith branched type galactosyllipid derivatives to targetasialoglycoprotein (galactose) receptors, which are exclusivelyexpressed on the surface of liver cells (Kato and Sugiyama, Crit. Rev.Ther. Drug Carrier Syst. 14:287 (1997); Murahashi et al., Biol. Pharm.Bull. 20:259 (1997)). Similarly, Wu et al., Hepatology 27:772 (1998),have shown that labeling liposomes with asialofetuin led to a shortenedliposome plasma half-life and greatly enhanced uptake ofasialofetuin-labeled liposome by hepatocytes. On the other hand, hepaticaccumulation of liposomes comprising branched type galactosyllipidderivatives can be inhibited by preinjection of asialofetuin (Murahashiet al., Biol. Pharm. Bull. 20:259 (1997)). Polyaconitylated human serumalbumin liposomes provide another approach for targeting liposomes toliver cells (Kamps et al., Proc. Nat'l Acad. Sci. USA 94:11681 (1997)).Moreover, Geho, et al. U.S. Pat. No. 4,603,044, describe ahepatocyte-directed liposome vesicle delivery system, which hasspecificity for hepatobiliary receptors associated with the specializedmetabolic cells of the liver.

In a more general approach to tissue targeting, target cells areprelabeled with biotinylated antibodies specific for a ligand expressedby the target cell (Harasym et al., Adv. Drug Deliv., Rev. 32:99(1998)). After plasma elimination of free antibody,streptavidin-conjugated liposomes are administered. In another approach,targeting antibodies are directly attached to liposomes (Harasym et al.,Adv. Drug Deliv. Rev. 32:99 (1998)).

Polypeptides and antibodies can be encapsulated within liposomes usingstandard techniques of protein microencapsulation (see, for example,Anderson et al., Infect. Immun. 31:1099 (1981), Anderson et al., CancerRes. 50:1853 (1990), and Cohen et al., Biochim. Biophys. Acta 1063:95(1991), Alving et al. “Preparation and Use of Liposomes in ImmunologicalStudies,” in Liposome Technology, 2nd Edition, Vol. III, Gregoriadis(ed.), page 317 (CRC Press 1993), Wassef et al., Meth. Enzymol. 149:124(1987)). As noted above, therapeutically useful liposomes may contain avariety of components. For example, liposomes may comprise lipidderivatives of poly(ethylene glycol) (Allen et al., Biochim. Biophys.Acta 1150:9 (1993)).

Degradable polymer microspheres have been designed to maintain highsystemic levels of therapeutic proteins. Microspheres are prepared fromdegradable polymers such as poly(lactide-co-glycolide) (PLG),polyanhydrides, poly(ortho esters), nonbiodegradable ethylvinyl acetatepolymers, in which proteins are entrapped in the polymer (Gombotz andPettit, Bioconjugate Chem. 6:332 (1995); Ranade, “Role of Polymers inDrug Delivery,” in Drug Delivery Systems, Ranade and Hollinger (eds.),pages 51-93 (CRC Press 1995); Roskos and Maskiewicz, “DegradableControlled Release Systems Useful for Protein Delivery,” in ProteinDelivery: Physical Systems, Sanders and Hendren (eds.), pages 45-92(Plenum Press 1997); Bartus et al., Science 281:1161 (1998); Putney andBurke, Nature Biotechnology 16:153 (1998); Putney, Curr. Opin. Chem.Biol. 2:548 (1998)). Polyethylene glycol (PEG)-coated nanospheres canalso provide carriers for intravenous administration of therapeuticproteins (see, for example, Gref et al., Pharm. Biotechnol. 10:167(1997)).

The antibody fragments described herein, including scFvs, Fabs,diabodies, etc. can be fused to an entity to extend their half-life.See, for example, Kubetzko S. et al. J Biol Chem 281:35186 (2006)[Modified p185Her2 (Her2)-specific scFv 4D5 and measured bindingaffinity, functional affinity, tumor distribution, and PK. Intumor-bearing mice, 20 kD PEGylation of the scFv monomer and dimerextended serum half-life and tumor distribution.]; Yang K et al. ProteinEngineering 16:761 (2003) [A 20 kD or 40 kD-PEGylated anti-TNF-a scFv(D2E7/Humira) retained affinity for target and pharmacological activity.Following iv administration in mice, 40 kD PEGylated scFv had a 200-foldincreased circulating half-life and an 800-fold increased AUC ascompared to the unmodified scFv.]; Chapman A P et al. Nature Biotech17:780 (1999). [Fab utilized in the experiments exploring affinities andhalf-life of random versus targeted PEG attachment and different sizesof the PEG molecule. Demonstrates site-specific attachment of PEGmoieties to the hinge region cysteine residue results in higher bindingaffinities for target as compared to randomly attached PEGs. Bindingaffinity as measured by Biacore analysis was identical for parent Ig andthe Fab molecules modified by attachment of 5 kD, 25 kD and branched 40kD PEGs. 25 kD and 40 kD-PEGylated Fabs had a comparable or increasedhalf-life and an AUC of 50-70% of that of the parent Ig molecule in miceand monkeys]; Muller D et al. J Biol Chem 282:12650 (2007). [Fused humanserum albumin (HSA) to several bispecific antibodies to carcinoembryonicantigen (CEA) and CD3 and compared activity and pharmacokinetics. scFv2,scDb, and taFv (constructs shown in paper) fused to HSA retained fullbinding capacity and activity. Half-life in serum of the HSA constructsincreased 5-11-fold and the AUC increased 6-7-fold compared to theirrespective unmodified parent molecule]; and Kipriyanov S M et al. J MolBiol 293:41 (1999). [Anti-CD3/CD-19 tandem diabody molecule demonstratedto have higher affinity, slower dissociation, higher stability in serum,higher in vivo stability, and longer in vivo half-life as compared to asingle chain Fv fragments and diabodies.]

The present invention also contemplates chemically modified polypeptideshaving binding IL-17 and IL-23 activity such as anti-IL-17A andIL-23/p19 antibodies, which a polypeptide is linked with a polymer, asdiscussed above.

Other dosage forms can be devised by those skilled in the art, as shown,for example, by Ansel and Popovich, Pharmaceutical Dosage Forms and DrugDelivery Systems, 5^(th) Edition (Lea & Febiger 1990), Gennaro (ed.),Remington's Pharmaceutical Sciences, 19^(th) Edition (Mack PublishingCompany 1995), and by Ranade and Hollinger, Drug Delivery Systems (CRCPress 1996).

As an illustration, pharmaceutical compositions may be supplied as a kitcomprising a container that comprises an antibody of the invention.Antibodies of the invention can be provided in the form of an injectablesolution for single or multiple doses, or as a sterile powder that willbe reconstituted before injection. Alternatively, such a kit can includea dry-powder disperser, liquid aerosol generator, or nebulizer foradministration of a therapeutic polypeptide. Such a kit may furthercomprise written information on indications and usage of thepharmaceutical composition. Moreover, such information may include astatement that the antibody composition is contraindicated in patientswith known hypersensitivity to IL-17 and IL-23.

A pharmaceutical composition comprising antibodies of the invention canbe furnished in liquid form, in an aerosol, or in solid form. Liquidforms, are illustrated by injectable solutions, aerosols, droplets,topological solutions and oral suspensions. Exemplary solid formsinclude capsules, tablets, and controlled-release forms. The latter formis illustrated by miniosmotic pumps and implants (Bremer et al., Pharm.Biotechnol. 10:239 (1997); Ranade, “Implants in Drug Delivery,” in DrugDelivery Systems, Ranade and Hollinger (eds.), pages 95-123 (CRC Press1995); Bremer et al., “Protein Delivery with Infusion Pumps,” in ProteinDelivery: Physical Systems, Sanders and Hendren (eds.), pages 239-254(Plenum Press 1997); Yewey et al., “Delivery of Proteins from aControlled Release Injectable Implant,” in Protein Delivery: PhysicalSystems, Sanders and Hendren (eds.), pages 93-117 (Plenum Press 1997)).Other solid forms include creams, pastes, other topologicalapplications, and the like.

Liposomes provide one means to deliver therapeutic polypeptides to asubject intravenously, intraperitoneally, intrathecally,intramuscularly, subcutaneously, or via oral administration, inhalation,or intranasal administration. Liposomes are microscopic vesicles thatconsist of one or more lipid bilayers surrounding aqueous compartments(see, generally, Bakker-Woudenberg et al., Eur. J. Clin. Microbial.Infect. Dis. 12 (Suppl. 1):S61 (1993), Kim, Drugs 46:618 (1993), andRanade, “Site-Specific Drug Delivery Using Liposomes as Carriers,” inDrug Delivery Systems, Ranade and Hollinger (eds.), pages 3-24 (CRCPress 1995)). Liposomes are similar in composition to cellular membranesand as a result, liposomes can be administered safely and arebiodegradable. Depending on the method of preparation, liposomes may beunilamellar or multilamellar, and liposomes can vary in size withdiameters ranging from 0.02 μm to greater than 10 μm. A variety ofagents can be encapsulated in liposomes: hydrophobic agents partition inthe bilayers and hydrophilic agents partition within the inner aqueousspace(s) (see, for example, Machy et al., Liposomes In Cell Biology AndPharmacology (John Libbey 1987), and Ostro et al., American J. Hosp.Pharm. 46:1576 (1989)). Moreover, it is possible to control thetherapeutic availability of the encapsulated agent by varying liposomesize, the number of bilayers, lipid composition, as well as the chargeand surface characteristics of the liposomes.

Liposomes can adsorb to virtually any type of cell and then slowlyrelease the encapsulated agent. Alternatively, an absorbed liposome maybe endocytosed by cells that are phagocytic. Endocytosis is followed byintralysosomal degradation of liposomal lipids and release of theencapsulated agents (Scherphof et al., Ann. N.Y. Acad. Sci. 446:368(1985)). After intravenous administration, small liposomes (0.1 to 1.0μm) are typically taken up by cells of the reticuloendothelial system,located principally in the liver and spleen, whereas liposomes largerthan 3.0 μm are deposited in the lung. This preferential uptake ofsmaller liposomes by the cells of the reticuloendothelial system hasbeen used to deliver chemotherapeutic agents to macrophages and totumors of the liver.

The reticuloendothelial system can be circumvented by several methodsincluding saturation with large doses of liposome particles, orselective macrophage inactivation by pharmacological means (Claassen etal., Biochim. Biophys. Acta 802:428 (1984)). In addition, incorporationof glycolipid- or polyethelene glycol-derivatized phospholipids intoliposome membranes has been shown to result in a significantly reduceduptake by the reticuloendothelial system (Allen et al, Biochim. Biophys.Acta 1068:133 (1991); Allen et al., Biochim. Biophys. Acta 1150:9(1993)).

Liposomes can also be prepared to target particular cells or organs byvarying phospholipid composition or by inserting receptors or ligandsinto the liposomes. For example, liposomes, prepared with a high contentof a nonionic surfactant, have been used to target the liver (Hayakawaet al, Japanese Patent 04-244,018; Kato et al., Biol. Pharm. Bull.16:960 (1993)). These formulations were prepared by mixing soybeanphosphatidylcholine, α-tocopherol, and ethoxylated hydrogenated castoroil (HCO-60) in methanol, concentrating the mixture under vacuum, andthen reconstituting the mixture with water. A liposomal formulation ofdipalmitoylphosphatidylcholine (DPPC) with a soybean-derivedsterylglucoside mixture (SG) and cholesterol (Ch) has also been shown totarget the liver (Shimizu et al, Biol. Pharm. Bull. 20:881 (1997)).

Alternatively, various targeting ligands can be bound to the surface ofthe liposome, such as antibodies of the present invention, antibodyfragments, carbohydrates, vitamins, and transport proteins. For example,liposomes can be modified with branched type galactosyllipid derivativesto target asialoglycoprotein (galactose) receptors, which areexclusively expressed on the surface of liver cells (Kato and Sugiyama,Crit. Rev. Ther. Drug Carrier Syst. 14:287 (1997); Murahashi et al.,Biol. Pharm. Bull. 20:259 (1997)). Similarly, Wu et al., Hepatology27:772 (1998), have shown that labeling liposomes with asialofetuin ledto a shortened liposome plasma half-life and greatly enhanced uptake ofasialofetuin-labeled liposome by hepatocytes. On the other hand, hepaticaccumulation of liposomes comprising branched type galactosyllipidderivatives can be inhibited by preinjection of asialofetuin (Murahashiet al., Biol. Pharm. Bull. 20:259 (1997)). Polyaconitylated human serumalbumin liposomes provide another approach for targeting liposomes toliver cells (Kamps et al., Proc. Nat'l Acad. Sci. USA 94:11681 (1997)).Moreover, Geho, et al. U.S. Pat. No. 4,603,044, describe ahepatocyte-directed liposome vesicle delivery system, which hasspecificity for hepatobiliary receptors associated with the specializedmetabolic cells of the liver.

In a more general approach to tissue targeting, target cells areprelabeled with biotinylated antibodies specific for a ligand expressedby the target cell (Harasym et al., Adv. Drug Deliv. Rev. 32:99 (1998)).After plasma elimination of free antibody, streptavidin-conjugatedliposomes are administered. In another approach, targeting antibodiesare directly attached to liposomes (Harasym et al., Adv. Drug Deliv.Rev. 32:99 (1998)).

The antibodies of the invention can be encapsulated within liposomesusing standard techniques of protein microencapsulation (see, forexample, Anderson et al., Infect. Immun. 31:1099 (1981), Anderson etal., Cancer Res. 50:1853 (1990), and Cohen et al., Biochim. Biophys.Acta 1063:95 (1991), Alving et al. “Preparation and Use of Liposomes inImmunological Studies,” in Liposome Technology, 2nd Edition, Vol. III,Gregoriadis (ed.), page 317 (CRC Press 1993), Wassef et al., Meth.Enzymol. 149:124 (1987)). As noted above, therapeutically usefulliposomes may contain a variety of components. For example, liposomesmay comprise lipid derivatives of poly(ethylene glycol) (Allen et al.,Biochim. Biophys. Acta 1150:9 (1993)).

Degradable polymer microspheres have been designed to maintain highsystemic levels of therapeutic proteins. Microspheres are prepared fromdegradable polymers such as poly(lactide-co-glycolide) (PLG),polyanhydrides, poly(ortho esters), nonbiodegradable ethylvinyl acetatepolymers, in which proteins are entrapped in the polymer (Gombotz andPettit, Bioconjugate Chem. 6:332 (1995); Ranade, “Role of Polymers inDrug Delivery,” in Drug Delivery Systems, Ranade and Hollinger (eds.),pages 51-93 (CRC Press 1995); Roskos and Maskiewicz, “DegradableControlled Release Systems Useful for Protein Delivery,” in ProteinDelivery: Physical Systems, Sanders and Hendren (eds.), pages 45-92(Plenum Press 1997); Bartus et al., Science 281:1161 (1998); Putney andBurke, Nature Biotechnology 16:153 (1998); Putney, Curr. Opin. Chem.Biol. 2:548 (1998)). Polyethylene glycol (PEG)-coated nanospheres canalso provide carriers for intravenous administration of therapeuticproteins (see, for example, Gref et al., Pharm. Biotechnol. 10:167(1997)).

Other dosage forms can be devised by those skilled in the art, as shown,for example, by Ansel and Popovich, Pharmaceutical Dosage Forms and DrugDelivery Systems, 5^(th) Edition (Lea & Febiger 1990), Gennaro (ed.),Remington's Pharmaceutical Sciences, 19^(th) Edition (Mack PublishingCompany 1995), and by Ranade and Hollinger, Drug Delivery Systems (CRCPress 1996).

The present invention contemplates antagonists of IL-17 and IL-23 andmethods and therapeutic uses comprising an such antagonists as describedherein. Such compositions can further comprise a carrier. The carriercan be a conventional organic or inorganic carrier. Examples of carriersinclude water, buffer solution, alcohol, propylene glycol, macrogol,sesame oil, corn oil, and the like.

The invention is further illustrated by the following non-limitingexamples.

EXAMPLES Example 1 Binding of Human IL-17 to Human IL-17R

A) Binding of Biotinylated IL-17 to Cells Transfected with the CognateIL-17 Receptor (IL-17R)

Baby Hamster Kidney (BHK) cells that had been transfected withexpression vectors encoding human IL-17R (polynucleotide shown in SEQ IDNO:7; polypeptide shown in SEQ ID NO:8) were assessed for their abilityto bind biotinylated human IL-17. Cells were harvested with versene,counted and diluted to 10⁷ cells per ml in staining media (SM), whichwas HBSS plus 1 mg/ml bovine serum albumin (BSA), 10 mM Hepes, and 0.1%sodium azide (w/v). Biotinylated human IL-17 (SEQ ID NO:2) wereincubated with the cells on ice for 30 minutes at variousconcentrations. After 30 minutes, excess cytokine was washed away withSM and the cells were incubated with a 1:100 dilution of streptavidinconjugated to phycoerythrin (SA-PE) for 30 minutes on ice. Excess SA-PEwas washed away and cells were analyzed by flow cytometry. The amount ofcytokine binding was quantitated from the mean fluorescence intensity ofthe cytokine staining. Results demonstrate that human IL-17 binds tohuman IL-17R-transfected cells with high affinity.

B) Inhibition of Specific Binding of Biotinylated Human IL-17 withUnlabeled Cytokine

Binding studies were performed as discussed above, but excess unlabeledhuman IL-17 was included in the binding reaction, as well as excessunlabeled IL-17B, IL-17C, IL-17D, IL-17E and IL-23. In studies with BHKcells, the amount of unlabeled cytokine was varied over a range ofconcentrations and we find that addition of unlabeled human IL-17competed for binding of human IL-17 to human IL-17R-transfected cell,indicating that human IL-17 specifically binds to human IL 17R; theother IL-17 family cytokines tested (B, C, D, E, and F) were not able tocompete for binding, further supporting the specificity of IL-17 forIL-17R and the use of this assay for testing antagonistic binding ofIL-17 with an antibody.

C) Inhibition of Specific Binding of Biotinylated Human IL-17 with anAnti-Human IL-17 Antagonist

Binding studies were performed as discussed above, except that a rangeof concentrations of an antagonist of the present invention (i.e. anantibody) to human IL-17 was included in the binding reactions. We findthat the anti-human IL-17 antibody inhibits binding of human IL-17 tohuman IL-17R-transfected BHK cells, indicating that the anti-human IL-17antibody was effective at blocking the binding of IL-17 to its receptor.Isotype-matched negative control antibodies and antibodies to IL-17cytokine family members other than IL-17 were unable to block thebinding of human IL-17 to IL-17RA-transfected cells, indicating that theaction of the anti-human IL-17 antibody was specific for human IL-17.

Example 2 Murine Nih3t3 Cells Respond to Human IL-17

A) Cell Plating and kz142 Adenovirus Reporter Infection

Nih3t3 cells, derived from mouse fibroblasts were plated at 5000cells/well in solid white, cell culture coated 96 well plates, (Cat.#3917. Costar) using DMEM/10% FBS, containing glutamine and amended withpyruvate and cultured overnight at 37° C. and 5% CO2. On this secondday, the plating media was removed and Kz142 adenovirus particles at amultiplicity of infection of 5000 particles/cell were prepared inDMEM/1% FBS, containing glutamine and amended with pyruvate and culturedovernight at 37° C. and 5% CO2.

B) Luciferase Assay Measuring IL-17 Activation of Kz142 AdenovirusReporter Infected Nih3t3 Cells

Following the overnight incubation with the adenovirus particlereporter, human IL-17 ligand treatments were prepared in “serum-free”media, amended to 0.28% BSA. The adenovirus particles and media wereremoved and the appropriate ligand doses were given in triplicates.Incubation at 37° C. and 5% CO2 was continued for 4 hours, after whichthe media was removed, cells lysed for 15 minutes and mean fluorescenceintensity (MFI) measured using the luciferase assay system and reagents.(Cat. #e1531 Promega. Madison, Wis.) and a Microplate luminometer.Activity was detected at concentrations ranging from 0.1-1000 ng/mLhuman IL-17, generating EC50 values of about 50 ng/L. These data suggestthat nih3t3 cells carry receptors for human IL-17 and that IL-17activates the NfKb/Ap-1 transcription factor, thus providing anappropriate cell line for testing IL-17-mediated activity and use of anantibody to augment this activity.

Example 3 Murine Nih3t3 Cells Express Human IL-17 Receptor (IL-17R)

RTPCR analysis of nih3t3 RNA demonstrated that these cells are positivefor human IL-17R, consistent with their nfkb/ap1 response to human IL-17being mediated through this receptor.

A) Mouse IL-17R PCR

First strand cDNA was prepared from total RNA isolated from nih3t3 cellsusing standard methods. PCR was applied using hot start polymerase andthe manufacturer's recommendations (Qiagen, Valencia, Calif.) usingsense primer, zc38520 (SEQ ID NO:9) and antisense primer, zc 38521 (SEQID NO:10) and 35 cycles of amplification. Agarose gel electrophoresisrevealed a single, robust amplicon of the expected, 498 bp size.

Example 4 Creation of a Stable Nih3t3 Assay Clone Expressing theap1/nfkb Transcription Factor

The murine nih3t3 cell line described above was stably transfected withthe kz142 ap1/nfkb reporter construct, containing a neomycin-selectablemarker. The Neo resistant transfection pool was plated at clonaldensity. Clones were isolated using cloning rings and screened byluciferase assay using the human IL-17 ligand as an inducer: Clones withthe highest mean fluorescence intensity (MFI) (via ap1/NfkB luciferase)and the lowest background were selected. A stable transfectant cell linewas selected and called nih3t3/kz142.8.

Example 5 Inhibition of Activation by Human IL-17 in Murine Nih3t3 CellsUsing an Antagonist to Human IL-17

Antibodies or other IL-17A neutralizing entities to human IL-17 wereused as antagonists of human IL-17 activation of ap1/nfkb elements in aluciferase assay. In this assay, anti-human IL-17 antibodies orneutralizing entities inhibit EC50 levels of human IL-17-mediatedap1/nfkb activation in the murine nih3t3/kz142.8 assay cell line. Forhighly effective antibodies, when used at approx. 10 μg/mLconcentration, the antibody completely neutralized activity induced byhuman IL-17, with the inhibition of activity decreasing in a dosedependent fashion at the lower concentrations. An isotype-matchednegative control mAb, tested at the concentrations described above,provided no inhibition of activity. These results demonstrate thatantibodies and other neutralizing entities to IL-17 are able toantagonize the activity of the pro-inflammatory cytokines, IL-17.Inhibition in this assay was not observed when anti-human antibodies toother ligand members of the IL-17 family, besides IL-17, were addedinstead of the anti-human IL-17 antibody or neutralizing entities. Table6 below shows representative example data for the ability ofneutralizing IL-17A antagonist positive controls, and anti-IL-17Aneutralizing entities described herein. The data demonstrate that theseneutralizing entities are able to reduce the activity induced by humanIL-17A.

TABLE 6 IC50 (nM) IL17A poly Ab 2 IL-17RA-Fc 1.3 IL-17A Fab cluster idClone c87 (SQ7) M7.19 E7 7.2 c86 (SQ7) M7.19 D10 7.7 c97 (SQ7) M7.20 G628 c95 (SQ7) M7.20 E5 105 c100 (SQ7) M7.24 G6 31 c99 (SQ7) M7.24 E8 84.0c83 (SQ7) M7.19 F4 100.0 c98 (SQ7) M7.24 E5 >130 c88 (SQ7) M7.24 A5 96.0c94 (SQ7) M7.20 C10 >130 c96 (SQ7) M7.20 F11 37.0 c90 (SQ7) M7.20 A9113.0 IL-17A scFv c222.1 M7.76_C08 33

Example 6 Neutralization of IL-17 Activity by an Anti-Human IL-17Antibody

Using a cell-based neutralization assay, a purified anti-human IL-17antibody was added as a serial dilution, for example, at 10 μg/ml, 5μg/ml, 2.5 μg/ml, 1.25 μg/ml, 625 ng/ml, 313 ng/ml, 156 ng/ml and 78ng/ml. The assay plates were incubated at 37° C., 5% CO₂ for 4 days atwhich time Alamar Blue (Accumed, Chicago, Ill.) was added at 20 μl/well.Plates were again incubated at 37° C., 5% CO₂ for 16 hours. This assayis able to demonstrate that the purified anti-human monoclonal antibodywas able neutralize signaling of human IL-17. For highly effectiveantibodies, when used at approx. 10 μg/mL concentration, the antibodycompletely neutralizes proliferation induced by human IL-17, with theinhibition of proliferation decreasing in a dose dependent fashion atthe lower concentrations. An isotype-matched negative control mouse mAb,tested at the concentrations described above, was expected to provide noinhibition of proliferation of either cytokine. These results furtherdemonstrate that antibodies to IL-17 could indeed antagonize theactivity of the pro-inflammatory ligands, IL-17. Inhibition in thisassay was not observed when anti-human antibodies to other ligandmembers of the IL-17 family, besides IL-17, are added instead of theanti-human IL-17 antibody, thus demonstrating IL-17A specificity.

Example 7 A Single Neutralizing Entity to IL-17 and IL-23 DecreasesInflammation and Inflammatory Mediators in an Ex Vivo Multiple SclerosisModel

Multiple sclerosis is a complex disease that is thought to be mediatedby a number of factors, including the presence of lymphocytic andmononuclear cell inflammatory infiltrates and demyelination throughoutthe CNS. Microglia are macrophage-like cells that populate the centralnervous system (CNS) and become activated upon injury or infection.Microglia have been implicated as playing critical roles in various CNSdiseases including multiple sclerosis, and may be used to studymechanism(s) of initiation, progression, and therapy of the disease(Nagai et al. Neurobiol. Dis. 8:1057 (2001); Olson et al. J NeurosciMethods 128:33 (2003)). Immortalized human microglial cell lines and/orestablished human astroglia cell lines can, therefore, be used to studysome of the effects of inflammatory mediators on these cell types andtheir potential for neutralization. Inflammatory mediators (includingbut not limited to IL-1b, IL-6, IL-8, IL-12, IL-13, IL-15, IL-17 A andF, IL-18, IL-23, TNF-a, IFN-g, MIP family members, RANTES, IP-10, MCP-1,G- and GM-CSF, etc.) can contribute to the symptoms and pathologyassociated with MS by way of their effect(s) on activating inflammatorypathways and downstream effector cells.

In order to evaluate the pro-inflammatory actions of IL-17 and IL-23,and the ability of an antagonist to these activities, such as moleculesthat bind IL-17 and IL-23, either singly or together, or antibodiesthereto including the IL-17/IL-23 antibodies of the present invention toneutralize or decrease these effects, cultured glial cells may betreated with one of the following: vehicle; rhIL-17; rhIL-23; or any ofthe compounds known to induce an inflammatory response by microglialand/or astroglia cells (e.g. LPS, other TLR agonists, inflammatorycytokines, etc.) In addition, these are treated with or without anantagonist of either IL-17 or IL-23, alone or in combination. Aftervarying times in culture (from 1 h to several days), supernatants andcells are collected and analyzed for levels and/or expression ofinflammatory mediators, including those listed above. Levels ofinflammatory cytokines and chemokines are elevated in the presence ofrhIL-17 and/or IL-23 and/or other inflammatory stimulants compared tocultures treated with vehicle alone. The addition of antagonists toIL-17 and/or IL-23 activity, such as the antibodies of the presentinvention markedly reduces the production and expression of inflammatorymediators, and thus, would expect to be efficacious in inflammatoryaspects associated with human multiple sclerosis.

Example 8 Neutralizing Entities to IL-1.7 and IL-23 Decreases DiseaseIncidence and Progression in Mouse Experimental AllergicEncephalomyelitis (EAE) as a Model of Multiple Sclerosis A) MouseAllergic Encephalomyelitis EAE Model

To study mechanism and evaluate the effects of potential therapies formultiple sclerosis, the animal model of experimental autoimmuneencephalomyelitis (EAE) is commonly used. For the relapsing-remittingEAE model, 9 to 10 week old female SJL mice (Jackson or Charles RiverLabs) were immunized subcutaneously with proteolipid peptide (PLP)emulsified in complete Freund's adjuvant, and with intravenous pertussistoxin. Within approximately 6 to 23 days, animals begin to show symptomsof weight loss and paralysis that are characteristic of this model. Theextent of disease is evaluated daily in the mice by taking their bodyweights and assigning a clinical score (0-8) to each mouse, as detailedbelow. The typical pattern of disease symptoms in immunized, butotherwise untreated mice, is one of weight loss and paralysis, followedby a period of disease symptom remission, and a subsequent relapse ofdisease symptoms. A pattern of relapses and remissions of diseasesymptoms ensues, which is also found in humans with this type ofmultiple sclerosis, known as relapsing-remitting disease. Chronicprogressive and secondary progressive multiple sclerosis are alsotargeted indications for this therapeutic combination of IL-17 andIL-23/p19 neutralizing antibodies or a single neutralizing entity suchas a bispecific antibody or scFV as described in this invention. Theselatter types of multiple sclerosis are tested in a similar manner usingMOG35-55 peptide in C57BL/6 mice, instead of PLP in SJL mice.

Neutralizing monoclonal antibodies to mouse IL-17 and IL-23p19 wereadministered separately or as a therapeutic combination, duringremission from the first peak of EAE disease symptoms. The antibodieswere delivered as intraperitoneal injections every other day, or as asimilar dosing regimen. Groups receive either 25, 50 or 100 μg of eachantibody, alone or as a therapeutic combination, per animal per dose,and control groups receive the vehicle control, PBS (Life Technologies,Rockville, Md.) or antibody isotype control.

B) Monitoring Disease

Animals can begin to show signs of paralysis and weight loss betweenapproximately 6 and 23 days following PLP or MOG35-55 immunizations.Most animals develop symptoms within 11-17 days of the immunizations,but some may show symptoms sooner or later than this.

All animals are observed, weighed, and assigned a clinical score dailyto assess the status of disease.

C) Clinical Score

0=Normal; healthy.

1=slight tail weakness (tip of tail does not curl and)

2=tail paralysis (unable to hold tail upright)

3=tail paralysis and mild waddle

4=tail paralysis and severe waddle

5=tail paralysis and paralysis of one limb

6=tail paralysis and paralysis of any 2 limbs

7=tetraparesis (all 4 limbs paralysed)

8=moribund or dead

Blood is collected throughout the experiment to monitor serum levels ofcytokine and levels of other mediators of disease. At the time ofeuthanasia, blood was collected for serum, and brain and spinal cordcollected in 10% NBF for histology. In separate animals, tissues(including lymph nodes, brain, spinal cord, spleen, and others) wereharvested for the quantification of mRNA by TaqMan quantitativereal-time PCR.

D) Results

Groups of mice (n=13-15 each) receiving the therapeutic combination ofneutralizing monoclonal antibodies to IL-17 and IL-23/p19 werecharacterized by a significant (p<0.05) reduction in disease severity asevidenced by significant (p<0.05) reductions in clinical score and bodyweight loss compared to mice treated with PBS, either of the antibodiesalone at similar doses as those used in the combination, or isotypecontrol antibodies. Furthermore, the mice treated with the therapeuticantibody combination had a complete absence of disease relapse, whereasall other treatment groups had 35-85% of mice experiencing significantdisease relapse. Therefore, the delivery of the therapeutic combinationwas significantly more efficacious in reducing active disease andimportantly, in preventing disease relapse, than the delivery of eithermonoclonal antibody alone at similar doses. This is a very importantfinding since disease relapse is a hallmark of this disease model and ofrelapsing-remitting MS in humans. There was extensive infiltration ofinflammatory cells into the CNS parenchyma for mice treated with onlythe PBS or isotype control antibody. The greatest overall reduction ofinflammatory cell infiltrates in the CNS of mice were those treated withthe therapeutic combination of neutralizing monoclonal antibodies toIL-17 and IL23/p19. Treatment with these therapeutic antibodies alsoresulted in significant reductions in serum IL-6, IL-13, IL-17A, IL-23,G-CSF, and TNF-a concentrations compared to PBS-treated mice. Sampleswere collected at the same time point following peak of first diseaseonset (day 27) and after the same number of antibody doses (11 doses).Draining lymph nodes were harvested from the mice at this same timepoint and cultured for 24 h with PLP139-151. Mice treated with thetherapeutic antibody combination had a lower percentage of TNF-acontaining draining lymph node cells compared to other groups of mice.Thus, the significant reductions in disease severity and disease relapsein the mice treated with the antibody combination were associated withreductions in CNS inflammatory infiltrates and inflammatory cytokineproduction, suggesting a mechanism of action for the observed efficacy.

Taken together, these results indicate that the therapeutic combinationof IL-17 and IL23/p19 neutralizing antibodies is more efficacious in thetreatment of EAE as a model of human multiple sclerosis. The therapeuticcombination can reduce clinical disease symptoms and works at themolecular level to reduce inflammation, inflammatory infiltrates,inflammatory cytokines/chemokines, and other mechanisms known to beaffected in this manner.

Example 9 Cell-Based Bioassay to Evaluate Activity of a Anti-MouseIL-23/p19 to Mouse IL-23

In an effort to develop a bioassay for use in testing IL-23 neutralizingantibodies, an IL-3-dependent mouse cell line expressing human DCRS2(IL-23R, SEQ ID NO: 22) was tested with recombinant mouse IL-23 in aproliferation assay to determine whether the human DCRS2 receptor alongwith endogenously expressed mouse IL-12RB1 can bind mouse IL-23 andcause cell signaling and proliferation. Once the ability of mouse IL-23to bind human DCRS2 was established, a second proliferation assay wasrun with mouse IL-23 and a monoclonal antibody to mouse IL-23/p19 todetermine its ability to specifically neutralize mouse IL-23/p19.

A) Construction of BaF3 Cells Expressing Full-Length DCRS2

The BaF3 assay cell line was a previously utilized cell line and waschosen for use in these assays because it expresses human DCRS2. Apreviously utilized BaF3 cell line that does not express human DCRS2 wasselected for use as a negative control. The assay cell line wasconstructed by sequentially placing an expression vector (pZP7Z)containing human WSX1 and an expression vector (pZP7NX) containing humanDCRS2 into BaF3 cells. These expression vectors and subsequent celllines were built using the following steps.

B) Cloning of Full length DCRS2

In US patent application number WO 00/73451, Dowling L M et al describedan orphan Class I cytokine receptor designated DCRS2 (SEQ ID NO:22)possessing a very short cytoplasmic domain such that it did not appearto be competent to signal if bound by its cognate ligand. In an effortto discover additional isoforms possessing cytoplasmic domains withclassical signaling elements, a PCR, 3'rapid amplification of cDNA ends(RACE) cloning strategy was initiated.

In order to identify cDNA template sources containing DCRS2 transcript,a collection of cDNA libraries were screened by PCR using zc38188 (SEQID NO:11) and zc38247 (SEQ ID NO:12). These oligos were used in a PCRreaction at a concentration of 0.2 pmol per uL, with Advantage IIthermostable DNA polymerase and buffer (Clontech, Palo Alto, Calif.),dNTPs at 0.125 mM and 40 ng per uL of cDNA library plasmid template.Following a 3 minute preincubation at 94° C. known as a “hotstart”, thereactions underwent 30 thermal cycles of 94° C., 30 seconds and 68° C.,90 seconds. Reaction products were analyzed by TAE, agarose gelelectrophoresis and ethidium bromide staining, and observed from thefollowing cDNA libraries: human adrenal, human bone marrow, humanactivated CD3+ T cell, and human testis. These products were isolated,sequenced and confirmed to be DCRS2. As these DCRS2-containing cDNAtemplates were directionally cloned, plasmid libraries; 3′RACE could bedone using “nested” oligo primers complimentary to the plasmid sequencedownstream of the 3′ cDNA cloning site. Primary 3′RACE PCR reactionswere run using the same reaction components as above but with zc 38188(SEQ ID NO:11) and zc 26405 (SEQ ID NO:13) and the four, DCRS2-positivecDNA libraries as templates. The products of these reactions werepurified using a Qiaquick PCR purification kit, (Qiagen, Valencia,Calif.) following the manufacturer's recommended protocol. The purifiedPCR products underwent a secondary amplification using PCR primers, zc38248 (SEQ ID NO:14), and zc 5020 (SEQ ID NO:15) whose complimentarysequences lie within the expected DCRS2-plasmid PCR product obtainedwith zc 38188 (SEQ ID NO:11) and zc 38247 (SEQ ID NO:12), and thus areso called “nested primers”. The thermal cycling conditions of thesereactions began with a hot start preincubation and continued with 30cycles of 94° C., 30 seconds; 64° C., 20 seconds; 71° C., 70 seconds.Products were gel-purified and sequenced directly using the sameamplifying primers. Sequence analysis revealed an extended cytoplasmicdomain containing putative box 1, 2 and 3 STAT binding motifs. The riskof these sequences containing mutations randomly introduced by PCR withAdvantageII over 60 cycles of amplification prompted repeating the PCRsusing a thermostable polymerase known to have higher fidelity and thus alower mutation rate.

In this second round of 3′ RACE, the 5′ primers were complimentary tovector sequence upstream of the cDNA cloning site while the 3′ primerswere designed based upon the sequence obtained from the 3′RACE productsdescribed above. Eight identical reactions were set up and run with arange of temperatures, 66-57° C., in the annealing step of the thermalcycle. Turbo Pful polymerase (Stratagene, LaJolla, Calif.) and itsbuffer along with dNTPs at 0.125 mM, 40 ng per uL human adrenal cDNAtemplate, zc 14063 (SEQ ID NO:16) and zc 38777 (SEQ ED NO:17) after ahotstart went through 30 thermal cycles of: 94° C., 30 seconds;annealing 35 seconds; 72° C., 130 seconds. The products of thesereactions were purified away from unincorporated primers using aQiaquick PCR purification kit, (Qiagen, Valencia, Calif.).

These products were then utilized as templates in a “nested” PCRreaction. Five identical, parallel reactions were assembled as above foreach template using zc 38776 (SEQ ID NO:18) and zc38188 (SEQ ID NO:11)with each reaction varying slightly in the annealing temperatures, 59,62, 63.5, 65, 66° C. A DNA product of approximately 1800 bp wasvisualized in all reactions that utilized a template originating from a57° C. primary reaction. by ethidium bromide staining following TAEagarose gel electrophoresis.

To facilitate the cloning of this PCR product into a mammalianexpression vector prior to sequencing, restriction sites were addedthrough the use of oligonucleotides and a third round of PCR with TurboPfu. Zc 38776 (SEQ ID NO:18) which contains an XhoI site, was mixed withzc 38246 (SEQ ID NO:19) in addition to the dNTPs, polymerase, buffer and1800 bp PCR product obtained above and following a hot start, incubatedfor 30 cycles of 95° C., 30 seconds; 55° C., 40 seconds; 72° C., 150seconds. The sequence of zc38776 matches the 5′ end of zc38188 but alsoincludes a BamHI site and Kozak concensus sequence. The resulting PCRproduct was purified using a Qiaquick mini-elute kit (Qiagen, Valencia,Calif.) and prepared for cloning into a mammalian expression vector bydigestion with BamHI and XhoI restriction enzymes for 90 minutes at 37°C. The mammalian expression vector pZP7NX was similarly digested withBamHI and XhoI to prepare it for acceptance of the full-length DCRS2cDNA. Both digestions underwent TAE gel electrophoresis, appropriatefragments were harvested and purified using a Qiaquick gel extractionkit (Qiagen, Valencia, Calif.) following the manufacturersrecommendations. Approximately 40 ng of BamHI/XhoI digested pZP7NX and10 ng of BamHI/XhoI digested DCRS2 were ligated together using 0.25 U T4DNA ligase (Invitrogen, Carlsbad, Calif.) and incubated at 22° C. for 4hours. One uL of the ligation reaction was then electroporated into 25uL of Electromax DH10b cells (Invitrogen, Carlsbad, Calif.) using aGenepulser electroporation apparatus (Biorad, Hercules, Calif.) set to2.3 Kv, 100 ohms and 25 uF. After plating serial dilutions of theelectroporation on LB-ampicillin agar plates, clones were isolated andconfirmed to be full-length with one silent mutation by sequenceanalysis (SEQ ID NO:22). A large scale plasmid prep was made and thisplasmid was utilized for various mammalian cell transfections.

BaF3, an IL-3-dependent prelymphoid cell line derived from murine bonemarrow (Palacios and Steinmetz, Cell 41: 727 (1985); Mathey-Prevot etal., Mol. Cell. Biol. 6:4133 (1986)), was maintained in complete media(RPMI medium; JRH Bioscience Inc., Lenexa, Kans.) supplemented with 10%heat-inactivated fetal calf serum, 2 ng/mL murine IL-3 (R&D,Minneapolis, Minn.), 2 mM L-glutamine (Gibco-BRL), and 1 mM sodiumpyruvate (Gibco-BRL).

BaF3 cells were prepared for electroporation by washing twice in RPMImedium (JRH Bioscience Inc., Lenexa, Kans.) and then resuspending inRPMI at a cell density of 10⁷ cells/ml. One mL of resuspended BaF3 cellswas mixed with 30 μg of the pZP7Z/h. WSX1 plasmid DNA and transferred toseparate disposable electroporation chambers (Gibco-BRL). The cells werethen given 2 serial shocks (800 lFad/300V; 1180 lFad/300V.) delivered byan electroporation apparatus (CELL-PORATOR™; Gibco-BRL, Bethesda, Md.).The electroporated cells were subsequently transferred to 20 mls ofcomplete media and placed in an incubator for 24 hours (37° C., 5% CO₂).The cells were then spun down and resuspended in 20 mLs of completemedia containing 240 μg/mL Zeocin (Invitrogen, Carlsbad, Calif.)selection in a T-75 flask to isolate the Zeocin resistant pool. Theresulting stable cell line was called BaF3/WSX1.

BaF3/WSX1 cells were prepared for electroporation by washing twice inRPMI medium (JRH Bioscience Inc., Lenexa, Kans.) and then resuspendingin RPMI at a cell density of 10⁷ cells/mL. One mL of resuspended BaF3cells was mixed with 30 μg of the pZP7NX_DCRS2 plasmid DNA andtransferred to separate disposable electroporation chambers (Gibco-BRL).The cells were then given 2 serial shocks (800 lFad/300V; 1180lFad/300V.) delivered by an electroporation apparatus (CELL-PORATOR™;Gibco-BRL, Bethesda, Md.). The electroporated cells were subsequentlytransferred to 20 mls of complete media containing 240 μg/mL Zeocin(Invitrogen, Carlsbad, Calif.) and placed in an incubator for 24 hours(37° C., 5% CO₂). The cells were then spun down and resuspended in 20mLs of complete media containing 1×G418 (Gibco-BRL, Bethesda, Md.) and240 μg/mL Zeocin (Invitrogen, Carlsbad, Calif.) selection in a T-75flask to isolate the G418 resistant pool. The resulting stable cell linewas called. BaF3 WSX1/DCRS2.

C) Construction of Baf3/KZ134 Cell Line

The negative control cell line, BaF3/KZ134, was constructed using thefollowing steps. The KZ134 plasmid was constructed with complementaryoligonucleotides ZC12749 (SEQ ID NO:20) and ZC12748 (SEQ ID NO:21) thatcontain STAT transcription factor binding elements from 4 genes, whichincludes a modified c-fos Sis inducible element (m67SIE, or hSIE)(Sadowski, et al., Science 261: 1739 (1993)) the p21 SIE1 from the p21WAF1 gene (Chin et al., Science 272: 719 (1996)), the mammary glandresponse element of the γ-casein gene (Schmitt-Ney et al., Mol. Cell.Biol. 11:3745 (1991)), and a STAT inducible element of the Fcγ RI gene,(Seidel et al., Proc. Natl. Acad. Sci. 92:3041 (1995)). Theseoligonucleotides contain Asp718-XhoI compatible ends and were ligated,using standard methods, into a recipient firefly luciferase reportervector with a c-fos promoter (Poulsen et al., J. Biol. Chem. 273:6229(1998)) digested with the same enzymes and containing a neomycinselectable marker. The KZ134 plasmid was used to stably transfect BaF3cells, using standard transfection and selection methods, to make theBaF3/KZ134 cell line.

D) Alamar Blue Proliferation Assay to Determine Species Specificity ofIL-23 on BaF3 Cells Expressing Human DCRS2

To determine whether human DCRS2 and mouse IL-12RB1 endogenouslyexpressed in murine BaF3 cells can bind mouse IL-23, and perhaps humanIL-23, an Alamar Blue proliferation assay was run. Recombinant humanIL-23 (R&D Systems, Cat. #1290-IL) and mouse IL-23 (R&D Systems, Cat.#1887-ML) were run at concentrations of 200, 100, 50, 25, 12.5, 6.3, and3.1 ng/mL. A positive control of mouse IL-3 was run at concentrations of20, 10, 5, 2.5, 1.25, 0.6, 0.3, and 0.15 pg/mL. Negative controls wererun in parallel using mouse IL-3-free media only. Samples were platedinto 96-well flat-bottomed plates (Bectin-Dickinson, Franklin Lakes,N.J.) in a volume of 100 μL. The cells were washed 3 times in IL-3 freemedia and counted using a hemocytometer. Cells were resuspended inIL-3-free media and plated at a concentration of 5000 cells per well in100 μL into the plate containing the samples for total well volume of200 μL. The assay plates are incubated at 37° C., 5% CO₂ for 3 days atwhich time Alamar Blue (Accumed, Chicago, Ill.) is added at 20 μL/well.Alamar Blue gives a fluorometric readout based on number of live cells,and thus is a direct measurement of cell proliferation in comparison toa negative control. Plates are again incubated at 37° C., 5% CO₂ for 24hours. Plates are read on the Wallac 1420 microplate reader (PerkinElmerLife Sciences, Boston, Mass.) at wavelengths 544 (Excitation), and 590(Emission).

Results show that both human and mouse IL-23 cause similar proliferativeresponses on the BaF3 WSX1/DCRS2 cell line. Human and mouse IL-23 werenegative on the BaF3/KZ134 cell line, which shows that the response islimited to the presence of DCRS2 expression. A second BaF3 cell lineexpressing human DCRS2 was also tested and was found to proliferate inresponse to human and mouse IL-23, similar to the BaF3 WSX1/DCRS2 cellline.

E) Alamar Blue Proliferation Assay to Determine Effects of aNeutralizing Antibody to Mouse IL-23/p19 on BaF3 Cells Expressing HumanDCRS2

Another proliferation assay was run to test the ability of an antibodyto neutralize mouse IL-23. The antibody tested was rat anti-mouseIL-23/p19 clone G23-8 (Cat. #16-723285, eBioscience, San Diego, Calif.).Mouse IL-23 concentrations of 10, 5, 2.5, 1.25, 0.6, 0.3, 0.15, and 0.08ng/mL were run. In order to determine whether the anti-IL-23/p19antibody was specific for the p19 sub-unit and did not cross-react withthe IL-12/23p40 subunit, a range of recombinant mouse IL-23p40concentrations (up to 200 ng/mL) was also tested. Concentrations of theanti-mouse IL-23/p19 antibody were run at 10, 5, 2.5, 1.25, 0.6, 0.3,0.15, and 0.08 ug/mL, and a negative control antibody, Rat IgG1anti-mouse CD115 (Cat. #MCA1848, Serotec, Raleigh, N.C.) was also run atthe same dilutions as the anti-mouse IL-23/p19 antibody. Mouse IL-23 wasadded to each well containing either the anti-mouse IL-23/p19 antibodyor mouse CD115 antibody for a final concentration of 1.5 ng/mL mouseIL-23. Wells without antibody were also set up with mouse IL-23 at 1.5ng/mL, which is approximately 80% of the maximum IL-23 response. Apositive control of mouse IL-3 was run at concentrations of 20, 10, 5,2.5, 1.25, 0.6, 0.3, and 0.15 pg/mL. Negative controls were run inparallel using mouse IL-3-free media only. Samples were plated into96-well flat-bottomed plates (Bectin-Dickinson, Franklin Lakes, N.J.) ina volume of 100 μL.

The cells were washed 3 times in IL-3-free media and counted using ahemocytometer. Cells were resuspended in IL-3-free media and plated at aconcentration of 5000 cells per well in 100 μL into the plate containingthe samples for a final well volume of 200 pt. The assay plates wereincubated at 37° C., 5% CO₂ for 3 days at which time Alamar Blue(Accumed, Chicago, Ill.) was added at 20 μL/well. Alamar Blue gives afluorometric readout based on number of live cells, and thus is a directmeasurement of cell proliferation in comparison to a negative control.Plates were again incubated at 37° C., 5% CO₂ for 24 hours. Plates wereread on the Wallac 1420 microplate reader (PerkinElmer Life Sciences,Boston, Mass.) at wavelengths 544 (Excitation), and 590 (Emission).

The results show that the anti-mouse IL-23/p19 antibody neutralized themouse IL-23 proliferative response in a dose-dependent manner. There wasno neutralization of the recombinant mouse IL-12/IL-23p40, thusindicates that the anti-mouse IL-23/p19 antibody is specific for the p19subunit of IL-23. The CD115 antibody slightly inhibits the proliferativeresponse at the highest concentration only. These assays show that thehuman DCRS2 receptor can cause signaling with mouse IL-23 in a BaF3proliferation assay system and can be used to screen neutralizingantibodies to mouse IL-23. Furthermore, these results demonstrate thatthe anti-mouse IL-23/p19 antibody is specific to the p19 subunit ofmouse IL-23.

Example 10 Cell-Based Bioassay to Evaluate Neutralizing Activity of anAnti-Human IL-23/p19 to Human IL-23

In an effort to develop a bioassay for use in testing human IL-23/p19neutralizing antibodies, an IL-3-dependent mouse cell line expressinghuman DCRS2 (IL-23R, SEQ ID NO: 22) was tested with recombinant humanIL-23 in a proliferation assay to determine whether the human DCRS2receptor along with endogenously expressed mouse IL-12RB 1 can bindhuman IL-23 and cause cell signaling and proliferation. As shown inExample 9 above, the cell line and resulting bioassay was found to beappropriate for the testing of both mouse and human IL-23 activity andIL-23 neutralizing activity. Therefore, once the ability of human IL-23to bind human DCRS2 was established, as shown in that example, a secondproliferation assay was run with human IL-23 and a monoclonal antibodyto human IL-23/p19 to determine its ability and specificity toneutralize human IL-23/p19.

A) Construction of BaF3 Cells Expressing Full-Length DCRS2 andConstruction of Baf3/KZ134 Cell Line

The BaF3 assay cell line was a previously utilized cell line and waschosen for use in these assays because it expresses human DCRS2. Apreviously utilized BaF3 cell line that does not express human DCRS2 wasselected for use as a negative control. The assay cell line wasconstructed by sequentially placing an expression vector (pZP7Z)containing human WSX1 and an expression vector (pZP7NX) containing humanDCRS2 into BaF3 cells. These expression vectors and subsequent celllines were built using the steps outlined in Example 9. The negativecontrol cell line, BaF3/KZ134, was constructed using the steps outlinedin Example 9.

B) Alamar Blue Proliferation Assay to Determine Effects of aNeutralizing Antibody to Human IL-23/p19 on BaF3 Cells Expressing HumanDCRS2

Another proliferation assay was run to test the ability of an antibodyto neutralize human IL-23, via IL-23/p19. Recombinant human IL-23concentrations of 10, 5, 2.5, 1.25, 0.6, 0.3, 0.15, and 0.08 ng/mL wererun. In order to determine whether the anti-IL-23/p19 antibody wasspecific for the p19 subunit and did not cross-react with theIL-12/IL-23p40 subunit, a range of recombinant human IL-12concentrations (up to 200 ng/mL) was also tested. Concentrations of theanti-human IL-23/p19 antibody were run at 10, 5, 2.5, 1.25, 0.6, 0.3,0.15, and 0.08 ug/mL, and a negative control antibody was also run atthe same dilutions as the anti-human IL-23/p19 antibody. Human IL-23 wasadded to each well containing either the anti-human IL-23/p19 antibodyor negative control antibody for a final concentration of 1.5 ng/mLhuman IL-23. Wells without antibody were also set up with human IL-23 at1.5 ng/mL, which is approximately 80% of the maximum IL-23 response. Apositive control of human IL-3 was run at concentrations of 20, 10, 5,2.5, 1.25, 0.6, 0.3, and 0.15 pg/mL. Negative controls were run inparallel using human IL-3-free media only. Samples were plated into96-well flat-bottomed plates (Bectin-Dickinson, Franklin Lakes, N.J.) ina volume of 100 μL.

The cells were washed 3 times in IL-3-free media and counted using ahemocytometer. Cells were resuspended in IL-3-free media and plated at aconcentration of 5000 cells per well in 100 μL into the plate containingthe samples for a final well volume of 200 μL. The assay plates wereincubated at 37° C., 5% CO₂ for 3 days at which time Alamar Blue(Accumed, Chicago, Ill.) was added at 20 μL/well. Alamar Blue gives afluorometric readout based on number of live cells, and thus is a directmeasurement of cell proliferation in comparison to a negative control.Plates were again incubated at 37° C., 5% CO₂ for 24 hours. Plates wereread on the Wallac 1420 microplate reader (PerkinElmer Life Sciences,Boston, Mass.) at wavelengths 544 (Excitation), and 590 (Emission).

The results show that the anti-human IL-23/p19 antibody neutralizes thehuman IL-23 proliferative response in a dose-dependent manner. There wasno neutralization of the exogenously added recombinant human IL-12, thusindicating that the anti-human IL-23/p19 antibody is specific for thep19 subunit of IL-23. Therefore, these results demonstrate that thehuman DCRS2 receptor can cause signaling with human IL-23 in a BaF3proliferation assay system and can be used to screen neutralizingantibodies to mouse IL-23. Furthermore, these results demonstrate thatthe anti-human IL-23/p19 antibody is specific to the p19 subunit ofIL-23.

Example 11 Murine IL23/p19 and IL-17 mRNAs are Regulated in SelectTissues in a Murine Model of Experimental Allergic Encephalomyelitis(EAE) Compared to Non-Diseased Controls

Tissues were obtained from mice at the peak of disease onset in the PLPEAE model. The model was performed following standard procedures ofimmunizing female SJL mice with PLP139-151, as described in Example 8above, in the presence or absence of pertussis toxin, and includedappropriate unimmunized, non-diseased controls. Tissues that werecollected included brain, spinal cord and cervical lymph nodes. RNA wasisolated from all tissues using standard procedures. In brief, tissueswere collected and immediately frozen in liquid N2 and then transferredto −80° C. until processing. For processing, tissues were placed inQiazol reagent (Qiagen, Valencia, Calif.) and RNA was isolated using theQiagen Rneasy kit according to manufacturer's recommendations.Expression of murine IL23/p19 and IL-17 mRNAs were measured withmultiplex real-time quantitative RT-PCR method (TaqMan) and the ABIPRISM 7900 sequence detection system (PE Applied Biosystems). IL23/p19and IL-17 mRNA levels were normalized to the expression of the murinehypoxanthine guanine physphoribosyl transferase mRNA and determined bythe comparative threshold cycle method (User Bulletin 2; PE AppliedBiosystems). The primers and probe for murine IL23/p19 included forwardprimer 5′ gcctgagttctagtcagcagtg, reverse primer 5′ tggaggcttcgaaggatct,and probe cagcgcccccttctccgttcc. The primers and probe for murine IL-17included forward primer 5′ gctccagaaggccctcaga, reverse primer 5′agcttccctccgcattga, and probe ctctccaccgcaatgaagaccctga. The resultswere as follows:

1) Murine IL23/p19 mRNA expression was detected in all tissues testedincluding brain, spinal cord and cervical lymph nodes;

2) Murine IL23/p19 mRNA levels were increased approximately 2.2-fold inthe spinal cord of mice immunized with PLP compared to unimmunizedcontrols;

3) Murine IL-17 mRNA expression was detected at very low levels in thelymph nodes and below the level of detection in the brain and spinalcord of unimmunized, control mice;

4) Murine IL-17 mRNA levels were increased approximately 104-fold in thebrain tissue of mice immunized with PLP compared to unimmunizedcontrols;

5) Murine IL-17 mRNA levels were increased approximately 695-fold in thespinal cord of mice immunized with PLP compared to unimmunized controls;and

6) Murine IL-17 mRNA levels were increased approximately 1.9-fold in thecervical lymph nodes of mice immunized with PLP compared to unimmunizedcontrols.

Because there are significantly higher levels of both IL-17 andIL-23/p19 in the CNS of mice (i.e. as measured in the same mice) withPLP-induced relapsing-remitting EAE, this further supports the need toantagonize both of these cytokines in multiple sclerosis. Thus,antagonists of the present invention have a therapeutic advantage overIL-17 or IL-23 treatment alone.

Example 12 IL-17 and IL-23/p19 are Overexpressed in Tissues from Micewith Collagen Induced Arthritis (CIA) Compared to Tissue fromNon-Diseased Mice A) Experimental Protocol

Tissues were obtained from mice with varying degrees of disease in thecollagen-induced arthritis (CIA) model. The model was performedfollowing standard procedures of immunizing male DBA/1J mice withcollagen in complete Freund's adjuvant (CFA) in the tail, followed 3weeks later by similar immunizations, but with collagen in incompleteFreund's adjuvant (IFA). Non-diseased age- and gender-matched DBA/1Jmice were also included for comparison. Tissues isolated includedaffected paws. RNA was isolated from the tissues using standardprocedures. In brief, tissues were collected and immediately frozen inliquid N2 and then transferred to −80° C. until processing. Forprocessing, tissues were placed in Qiazol reagent (Qiagen, Valencia,Calif.) and RNA was isolated using the Qiagen Rneasy kit according tomanufacturer's recommendations. Expression of murine IL-17 and IL-23/p19mRNA was measured with multiplex real-time quantitative RT-PCR methods(TaqMan) and the ABI PRISM 7900 sequence detection system (PE AppliedBiosystems). Murine IL-17 and IL-23/p19 mRNA levels were normalized tothe expression of murine hypoxanthine guanine physphoribosyl transferasemRNA and determined by the comparative threshold cycle method (UserBulletin 2: PE Applied Biosystems). The primers and probe for murineIL-17 and IL-23/p19 were the same as described in Example 11.

B) Results

Murine IL-17 and IL-23/p19 mRNA expression was detected in the tissuestested. Both IL-17 and IL-23/p19 mRNA were increased in the paws frommice in the CIA model of arthritis compared to tissues obtained fromnon-diseased controls. Murine IL-17 mRNA was increased in the pawsapproximately 8-fold in mice with more mild disease and approximately9.3-fold in mice with more severe disease compared to non-diseasedcontrols. Murine IL-23/p19 mRNA was increased in the paws approximately2.4-fold in mice with more mild disease and approximately 2.1-fold inmice with more severe disease compared to non-diseased controls.

Because there are significantly higher levels of both IL-17 andIL-23/p19 in the affected paws of mice (i.e. as measured in the samemice) with CIA, this further supports the need to antagonize both ofthese cytokines in rheumatoid arthritis.

Example 13 IL-17 and IL-23/p19 are Overexpressed in Tissues from Micewith DSS-Induced Colitis Compared to Tissue from Non-Diseased Mice A)Experimental Protocol

Tissues were obtained from mice in the dextran sodium sulfate (DSS)model of colitis. The model was performed following standard proceduresof administering 1.5-2.5% DSS in the drinking water of female C57BL/6mice for 5-7 days (acute protocol), followed by cycles of regular waterand DSS water (chronic protocol). Non-diseased age- and gender-matchedC57BL/6 mice were also included for comparison. Tissues isolatedincluded the descending colon and proximal colon. RNA was isolated fromthe tissues using standard procedures. In brief, tissues were collectedand immediately frozen in liquid N2 and then transferred to −80° C.until processing. For processing, tissues were placed in Qiazol reagent(Qiagen, Valencia, Calif.) and RNA was isolated using the Qiagen Rneasykit according to manufacturer's recommendations. Expression of murineIL-17 and IL-23/p19 mRNA was measured with multiplex real-timequantitative RT-PCR methods (TaqMan) and the ABI PRISM 7900 sequencedetection system (PE Applied Biosystems). Murine IL-17 and IL-23/p19mRNA levels were normalized to the expression of murine hypoxanthineguanine physphoribosyl transferase mRNA and determined by thecomparative threshold cycle method (User Bulletin 2: PE AppliedBiosystems). The primers and probe for murine IL-17 and IL-23/p19 werethe same as described in Example 11.

B) Results

Murine IL-17 and IL-23/p19 mRNA expression was detected in the tissuestested. Both IL-17 and IL-23/p19 mRNA were increased in the descendingand proximal colon from mice in the acute and chronic DSS colitis model,compared to tissues obtained from non-diseased controls. Murine IL-17mRNA was increased approximately 50-fold in the descending colon andapproximately 180-fold in the proximal colon of mice treated acutelywith DSS compared to non-diseased controls. With chronic DSS treatment,IL-17 mRNA levels were approximately 21- and 22-fold higher in thedescending and proximal colon, respectively, compared to non-diseasedcontrols. Murine IL-23/p19 mRNA was increased approximately 2-fold inthe descending colon and approximately 4.4-fold in the proximal colon ofmice treated acutely with DSS compared to non-diseased controls. Withchronic DSS treatment, IL-23/p19 mRNA levels were approximately 1.5- and2-fold higher in the descending and proximal colon, respectively,compared to non-diseased controls. Because there are significantlyhigher levels of both IL-17 and IL-23/p19 in the colons of mice (i.e. asmeasured in the same mice) with DSS colitis, this further supports theneed to antagonize both of these cytokines in inflammatory boweldisease.

Example 14 Bioassay for Neutralization of Human IL-23 Mediated IL-17Aand IL-17F Production in Murine Splenocytes

Recombinant human IL-23 (rhIL-23) induced the production of IL-17A andIL-17F in murine splenocytes. To evaluate antagonists to IL-23, weexamined the neutralization of IL-17A and IL-17F production in rhIL-23treated murine splenocytes. Antagonists to rhIL-23 are compared to thecommercial neutralizing antibody anti-IL-12p40 (Pharmingen, FranklinLakes, N.J.).

Experimental Protocol:

A single cell suspension of splenocytes were prepared from whole spleensharvested from either C57BL/6 or BALB/c mice. After red blood cell lysiswith ACK buffer (0.010 M KHCO3, 0.0001 M EDTA, 0.150 M NH4Cl),splenocytes were washed and resuspended in RPMI buffer (containing 1%non-essential amino acids, 1% Sodium Pyruvate, 2.5 mM HEPES, 1%L-glutamine, 0.00035% 2-mercaptoethanol, 1% Pen/Strep, 10% FCS and 50ng/ml human IL-2 (R&D Systems, Minneapolis, Minn.)). Cells were seededat 500,000 cells per well in a 96-well round bottom plate. In a separateplate, rhIL-23 at a concentration of 10 pM was pre-incubated for 30-90minutes at 37° C. with 3-fold serial dilutions of the antagonists listedin Table 7. Concentrations of the antagonists range from 0-343 nM. TheIL-23 ligand plus antagonists were then added to the splenocytes andincubated at 37° C., 5% CO2 for 24-72 hours. The supernatants werecollected and frozen at −80° C. until ready to process. The levels ofIL-17A and IL-17F protein in the supernatants were measured usingbead-based sandwich ELISAs. A commercial kit (Upstate, Charlottesville,Va.) was used to measure IL-17A protein. A bead-based ELISA developedin-house using an antibody to IL-17F (R&D) conjugated to a bead was usedto measure IL-17F. IC50 values for each antagonist were calculated asthe amount of antagonist needed to neutralize 50% of the activity ofrhIL-23.

Results:

In the presence of rhIL-23, the antagonists described in Table 7 wereefficacious at reducing IL-17A and IL-17F production with IC50 values of0.27-100.0 nM.

TABLE 7 IC50 values measured in the murine splenocyte assay. IC50 forIC50 for Cluster neutralizing neutralizing Antagonist number IL-17AIL-17F Anti-IL12p40 0.27 nM   0.4 nM  (Pharmingen) Anti-IL23p19 28 nM 30nM polyAb (R&D) IL23RA-Fc 10 nM 43 nM (ZGI) M7.12_B09 c41 (SQ7) 27 nM100 nM  (Fab) M7.36_B06 c305 (SQ22) 3.3 nM  14 nM (scFv)

Example 15 Plate-Based Binding Assay for IL-17A Materials and Methods:

Costar (#9018) 96-well plates were coated with 50 ul IL-17A (madein-house) or IL-17F (made in-house) at 2 ug/ml in 0.1M NaHCO₃, pH 9.6overnight at 4° C. The next day, plates were washed three times with0.1% Tween-20/PBS (PBST). Each well was filled with 350 ul of 2% milk(#170-6404, Bio-Rad)/PBST for one hour at RT for blocking. Assay plateswere then washed three times with PBST. Each well was filled with 50 ulof 2% milk/PBST, followed by the addition of 25 ul of Fab or scFvsupernatant. Wells were then mixed and then incubated for one hour atRT. Plates were washed three times with PBST. For Fab detection, 50 ulof (1:4000) anti-Human Fab specific pAb-HRP (#31482, Pierce) in 2%milk/PBST was added to each well for one hour at RT. For scFv detection,50 ul of (1:4000) anti-His tag mAb-HRP (Sigma, #A7058) in 2% milk/PBSTwas added to each well for one hour at RT. Plates were then washed threetimes with PBST. 50 ul of TMB (TMBW-1000-01, BioFX Laboratories) wasadded to each well to develop for 20-30 min, followed by the addition of50 ul of stop buffer (STPR-1000-01, BioFX Laboratories) to quench thereaction. Plates were then read at 450 nm on a plate reader. Wells thatshowed greater binding on the plate coated with IL-17A than on the platecoated with IL-17F were chosen for further analysis and manipulation.

Example 16 Plate-Based Binding Assay for IL-23 Materials and Methods:

Costar (#9018) 96-well plates were coated with 50 ul IL-23 (madein-house) or IL-12 (#219-IL/CF, R&D Systems) at 4 ng/ml in 0.1M NaHCO₃,pH 9.6, overnight at 4° C. The next day, plates were washed three timeswith 0.1% Tween-20/PBS (PBST). Each well was filled with 350 ul of 2%milk (#170-6404, Bio-Rad)/PBST for one hour at RT for blocking. Assayplates were then washed three times with PBST. Each well was filled with50 ul of 2% milk/PBST, followed by the addition of 25 ul of Fab or scFvsupernatant. Well were mixed and then incubated for one hour at RT.Plates were washed three times with PBST. For Fab detection, 50 ul of(1:4000) anti-Human Fab specific pAb-HRP (#31482, Pierce) in 2%milk/PBST was added to each well for one hour at RT. For scFv detection,50 ul of (1:4000) anti-His tag mAb-HRP (Sigma, #A7058) in 2% milk/PBSTwas added to each well for one hour at RT. Plates were washed threetimes with PBST. 50 ul of TMB (TMBW-1000-01, BioFX Laboratories) addedto each well to develop for 20-30 min, followed by the addition of 50 ulof stop buffer (STPR-1000-01, BioFX Laboratories) to quench thereaction. Plates were then read at 450 nm on a plate reader.

Wells that showed greater binding on the plate coated with the IL-23heterodimer than on the plate coated with the IL-12 heterodimer werechosen for further analysis and manipulation.

Example 17 Plate-Based Neutralization Assay for IL-17A

Materials and Methods:

Costar (#9018) 96-well plates were coated with 50 ul of anti-human IgGFcγ-specific antibody (#109-005-098, Jackson Immunology) at 1 ug/ml in0.1M NaHCO₃, pH 9.6 overnight at 4° C. The next day, plates were washedthree times with 0.1% Tween-20/PBS (PBST). 50 ul of IL-17RA (madein-house) at 0.25 ug/ml in PBS was added to each well, followed by aone-hour incubation at room temperature (RT). Plates were then washedthree times with PBST. Each well was filled with 350 ul of 2% milk(#170-6404, Bio-Rad)/PBST for one hour at RT for blocking. During plateblocking, adjacent plates were set up for pre-incubation of biotinylatedIL-17A with Fab or scFv supernatant. Pre-incubation plates were filledwith 75 ul of Fab supernatant, followed by the addition of 25 ul ofIL-17A (made in-house) at 0.10 ug/ml in 4% milk/PBST. Each well wasmixed and then incubated for 30 min at RT. Assay plates were then washedthree times with PBST, and the volume of the supernatant/IL-17A complextransferred from the pre-incubation plate to the assay plate, followedby a one-hour incubation at RT. Plates were then washed three times withPBST. 50 ul of (1:3000) Streptavidin-HRP (#21124, Pierce) in PBST addedto each well to incubate for one hour. Plates then washed three timeswith PBST. 50 ul of TMB (TMBW-1000-01, BioFX Laboratories) added to eachwell to develop for 20-30 min, followed by the addition of 50 ul of stopbuffer (STPR-1000-01, BioFX Laboratories) to quench the reaction. Plateswere then read at 450 nm on a plate reader.

Example 18 Plate-Based Neutralization Assay for IL-23

Materials and Methods:

Costar (#9018) 96-well plates were coated with 50 ul of anti-human IgGFcγ-specific antibody (#109-005-098, Jackson Immunology) at 1 ug/ml in0.1M NaHCO3, pH 9.6 overnight at 4° C. The next day, plates were washedthree times with 0.1% Tween-20/PBS (PBST). 50 ul of IL-23R(#1400-IR-050, R&D Systems) at 0.25 ug/ml in PBS was added to each well,followed by a one-hour incubation at room temperature (RT). Plates werethen washed three times with PBST. Each well was filled with 350 ul of2% milk (#170-6404, Bio-Rad)/PBST for one hour at RT for blocking.During plate blocking, adjacent plates were set up for pre-incubation ofbiotinylated IL-23 with Fab or scFv supernatant. Pre-incubation plateswere filled with 75 ul of Fab or scFv supernatant, followed by theaddition of 25 ul of IL-23 heterodimer or p19 (made in-house) at 0.10ug/ml in 4% milk/PBST. Each well was mixed and then incubated for 30 minat RT. Assay plates were then washed three times with PBST, and thevolume of the supernatant/IL-23 complex transferred from thepre-incubation plate to the assay plate, followed by a one-hourincubation at RT. Plates were then washed three times with PBST. 50 ulof (1:3000) Streptavidin-HRP (#21124, Pierce) in PBST added to each wellto incubate for one hour. Plates were then washed three times with PBST.50 ul of TMB (TMBW-1000-01, BioFX Laboratories) added to each well todevelop for 20-30 min, followed by the addition of 50 ul of stop buffer(STPR-1000-01, BioFX Laboratories) to quench the reaction. Plates werethen read at 450 nm on a plate reader.

Example 19 IL-17 and IL-23/p19 are Overexpressed in Tissues from MouseModel of T-Cell Adoptive Transfer Colitis Compared to Tissue fromNon-Diseased Mice T-Cell Adoptive Transfer Colitis Model

Adoptive transfer of naive T cells into minor histocompatibilitymismatched or syngeneic immunocompromised mice leads to development ofcolitis (Leach M W et al 1996, Powrie F et al, 1997) as well as skinlesions resembling psoriasis (Schon M P et al 1997, Davenport C M et al2002). Transplantation of as low as 0.2 million CD4+CD25− T cells fromBalb/C mice into immunocompromised C.B-17 SCID mice results in weightloss, hemoccult positive stool and development of skin lesions (thesymptoms in these mice vary from colony to colony). These symptomsnormally arise in mice between 7-10 weeks after transplantation.

This model of colitis has some similarities to human Crohn's disease andhas been used extensively to test efficacy of therapeutics for thisdisease in humans. For this experiment, mice (10 Balb/C females, 20 CB17SCID female) were obtained from CRL. Mice were on tap water starting onday −6. Spleens from 10 Balb/C mice will be collected. CD4+CD25− T-cellwill be collected from pooled spleen (see below for methods). CB17 SCIDmice will receive either 5×10̂5 or 7.5×10̂5 CD4+CD25− T-cells from spleenvia i.v. injection. All mice are weighed 3× week and carefully observedfor weight loss. When weight loss is observed, the Disease ActivityIndex (DAI) score [stool consistency, body weight, and blood in stool]are measured. Any animal with DAI score of 4 or body weight loss ofgreater than 20% are euthanized. There are no whole splenocytes control.

For LPS/IL-12 accelerated psoriasis, CB17 SCID mice will receive 5×105CD4+ CD25− T-cells from spleen via i.v. injection. On day 0, 7, and 14,mice are treated with Mug of LPS (from salmonella) and 10 ng of rm IL-12in 100 ul i.p. injection. All mice are weighed 3× week and carefullyobserved for weight loss. When ear thickening is observed, ear thicknessare measured 3× week. Mice are carefully monitored for signs ofpsoriasis (hair loss, scratching, alopecia, etc). Treatment groups wereas follows: Colitis: (Group I) received 0.5 mil CD4+CD25 cells, with a Nof 6 and (Group II) received 0.75 mil CD4+CD25 cells, with a N of 6.Psoriasis (Group III) received 0.5 mil CD4+CD25 cells, with a N of 4.

For histology, the distal part of small intestine and entire colon arecollected in 10% NBF then transfer to 70% ETOH after 24 hours. Samplesare submitted for histological evidence of colitis including presence ofgranuloma. For GEMS, MLN, distal part of small intestine, proximal colonand distal colon are collected and snap frozen in liquid nitrogen.

CD4+CD25− T-cell isolation: Balb/C mice are sacrificed by CO2asphyxiation and spleens are removed and single cell suspensions made.CD4 T cells are enriched by using a sterile magnetic enrichmentprotocol. CD4+CD25− T cells are further enriched using a sterilemagnetic enrichment protocol or by sorting using a cell sorter. Purityof T-cells are evaluated by flow.

Adoptive Transfer of CD4+CD25− T cells into SCID mice: ImmunocompromisedC.B-17 SCID mice were injected i.v. with either 0.5 or 0.75 millionenriched CD4+CD25− T cells on d0. Colitis animals were sacrificed. Micestarted to show sign of BW loss around day 15. On day 21, mice wereobserved with soft stool and diarrhea and were sacrificed. Mice werebled for serum via retro-orbital bleed then euthanized by cervicaldislocation. An entire intestine was removed and entire colon wasexcised for length measurement. The Colon was cut in half (proximal anddistal) and snap frozen. The ileum proximal to cecum was removed andsnap frozen. Fecal matter was carefully removed from all the intestinaltissues collected. Mesenteric LNs were collected and snap frozen. ForHistology (2 out of 6 animals from each group) an entire intestine wasremoved and flushed with PBS followed by 10% NBF. The entire intestine,intact, was fixed overnight in 10% NBA and transferred to 70% ETOH andsubmitted for histology.

Tissues were obtained from mice after onset of disease in a murine modelof T-cell adoptive transfer colitis. The model was performed followingstandard procedures of adoptive transfer of naive T cells into minorhistocompatibility mismatched or syngeneic immunocompromised mice andincluded appropriate immunocompromised, non-diseased controls. Tissuesthat were collected included the distal part of small-intestine,proximal colon, distal colon and mesenteric lymph node. RNA was isolatedfrom all tissues using standard procedures. In brief, tissues werecollected and immediately frozen in liquid N2 and then transferred to−800 C until processing. For processing, tissues were placed in Qiazolreagent (Qiagen, Valencia, Calif.) and RNA was isolated using the QiagenRneasy kit according to manufacturer's recommendations. Expression wasmeasured with multiplex real-time quantitative RT-PCR method (TaqMan)and the ABI PRISM 7900 sequence detection system (PE AppliedBiosystems). IL-17mRNA levels were normalized to the expression of themurine hypoxanthine guanine physphoribosyl transferase mRNA anddetermined by the comparative threshold cycle method (User Bulletin 2;PE Applied Biosystems). The primers and probe for murine IL-17A andIL-23p19 were the same as described in Example 11.

Results:

IL-17A mRNA:

-   -   In the distal colon, mice with colitis had levels of IL-17A mRNA        that were approximately 9000-fold greater than levels from        control mice (no colitis).    -   In the proximal colon, mice with colitis had levels of IL-17A        mRNA that were approximately 18,900-fold greater than levels        from control mice (no colitis).    -   In the small intestine, mice with colitis had levels of IL-17A        mRNA that were approximately 990-fold greater than levels from        control mice (no colitis).    -   In the mesenteric lymph nodes, mice with colitis had levels of        IL-17A mRNA that were approximately 175-fold greater than levels        from control mice (no colitis).    -   In all these samples, IL-17A mRNA was basically undetectable in        control mice.        IL-17F mRNA    -   In the distal colon, mice with colitis had levels of IL-17F mRNA        that were approximately 3.8-fold greater than levels from        control mice (no colitis).    -   In the proximal colon, mice with colitis had levels of IL-17F        mRNA that were approximately 7.4-fold greater than levels from        control mice (no colitis).    -   In the small intestine, mice with colitis had levels of IL-17F        mRNA that were approximately 4.8-fold greater than levels from        control mice (no colitis).    -   In the mesenteric lymph nodes, mice with colitis had levels of        IL-17F mRNA that were approximately 213-fold greater than levels        from control mice (no colitis).        IL-23p19 mRNA    -   In the small intestine, mice with colitis had levels of IL-23p19        mRNA that were approximately 2-fold greater than levels from        control mice (no colitis).    -   In the mesenteric lymph nodes, mice with colitis had levels of        IL-17A mRNA that were approximately 1.5-fold greater than levels        from control mice (no colitis).

Example 20 Effects of IL-17A and IL-23 on Lamina PropPria T Cells andMonocytes/Macrophages from Normal and Human IBD Samples

Dysregulated or sustained immune-mediated inflammation may contribute tothe symptoms and pathology associated with IBD by way of tissue damageor permanent skewing to inappropriate or prolonged immune responses.This model can determine the potential down-stream consequences ofexposure of disease-associated T cells and monocytes to IL-17A and II 23which may be present in the immediate environmental cytokine milieu ofthe intestinal tissue.

Therapeutics that would be efficacious in human IBD in vivo, would workin ex vivo models by inhibiting and/or neutralizing the productionand/or presence of inflammatory mediators (including but not limited toIL-1b, IL-4, IL-5, IL-6, IL-8, IL-12, IL-13, IL-15, IL-17 A and F,IL-18, IL-23, TNT-a, IFN-g, MIP family members, MCP-1, G- and GM-CSF,etc.).

In this model, T cells and monocytes/macrophages are isolated fromintestinal biopsy samples by carefully mincing biopsies with scissors inHBSS, treating with collagenase and Dispase II and incubating for 1 hrat 37° C. in a shaker. The cell suspension is filtered through nylonmesh to remove debris and cell clumps and washed multiple times in HBSS.T cells and macrophage/monocytes can be isolated using direct cellsorting or bead-depletion/enrichment protocols. Isolated cells areincubated in the presence of IL-17A and IL-23. This induces theproduction of inflammatory mediators by T cells andmonocytes/macrophages or results in skewing subsequent T cell responsesto highly pro-inflammatory responses. Comparisons between the types ofinflammatory mediators produced by cells from IBD patients and thosefrom cells of normal individuals can be made and might suggest that Tcells and monocyte/macrophages from IBD patients produce a morepro-inflammatory profile in the presence of IL-17A and IL-23. Theaddition of antibodies to IL-17A and IL-23p19 to neutralize theproduction of downstream inflammatory mediators may be efficacious inthe therapeutic treatment of patients with IBD.

Example 21 Efficacy of Antibodies that to Both IL-17A and IL-23 inIrritable Bowl Syndrome (“IBS”) CNS-Directed Pathogenesis

A model focusing on primary CNS-directed pathogenesis of IBS whichemploys stress stimuli to induce symptoms characteristic of TBS. Theneonatal psychosocial stress model mimics some clinical featuresassociated with IBS patients including visceral hyperalgesia, diarrheaand stress-sensitivity. Daily separation of the litter from theirmothers for 180 minutes each day during postnatal days 4-18 will resultin an alteration of maternal behaviour and significantly reduce times ofthe licking/grooming behaviour. The stress on the neonates results inpermanent changes in the CNS resulting in altered stress-inducedvisceral and somatic pain sensitivity. Colonic motor function inresponse to stress is enhanced in these animals and preliminary datashows evidence of increased intestinal permeability (Mayer et al., Eur.J. surg. Suppl. (587): 3-9 (2002)). Treatment with the antibodies of thepresent invention and subsequent analysis of colonic motor function,epithelial permeability and response to stress stimuli could determineefficacy in this animal model of IBS. Decreases in the incidence ofsymptoms following treatment with these inhibitors would suggestpotential efficacy in the treatment of IBS.

Example 22 Efficacy of Antibodies that Antagonize IL-17A and IL-23p19 inIrritable Bowl Syndrome (“IBS”) Primary Gut-Directed Inducers of Stress

This is a model focusing on primary gut-directed inducers of stress (ie.gut inflammation, infection or physical stress). Animal studies haveindicated that low-grade inflammation or immune activation may be abasis for altered motility, and/or afferent and epithelial function ofthe gut (Mayer et al., Eur. J. surg. Suppl. (587): 3-9 (2002)). In thismodel, daily colon irritation is produced in neonatal animals (days8-21) in the form of daily intracolonic injection of mustard oil.Mustard oil is a neural stimulant and has been shown to induce visceralhyperalgesia following intracolonic administration. This model mimicskey features of the IBS including visceral hypersensitivity andalteration in bowel habits. Animals also present with diarrhea orconstipation, a key feature of IBS patients (Mayer et al., Eur. J. surg.Suppl. (587): 3-9 (2002)); (Kimball et al., Am. J. Physiol.Gastrointest. Liver Pathol 288: G1266 (2005)). An antibody of thepresent invention could be delivered to determine changes in thedevelopment of symptoms associated with this model. Decreases in theincidence or magnitude of visceral hypersensitivity and altered gutmotility following therapeutic treatment with our inhibitors wouldsuggest a potential for these molecules to be efficacious in thetreatment of IBS.

Example 23 IL-17 NIH-3T3/huIL-17RCx4 Iκβ-α Bioassay

The NIH-3T3/KZ142.8/huIL-17RCx4 transfected cell line was generated asdescribed in WO 2005/123778, filed Jun. 10, 2005. On day oneNIH-3T3/KZ142.8/huIL-17RCx4 cells were plated out at 7,500 cells/well ingrowth media (DMEM with L-Glutamine plus 5% fetal bovine serum, 1%Sodium Pyruvate, 1 μM MTX) in 96-well, flat-bottom tissue cultureplates. On day two cells were switched to assay media (DMEM withL-Glutamine plus 0.1% BSA and 10 mM HEPES). On day three serialdilutions of human IL-17A (ZGI E. coli material and ZGI 293F B material)were made up in assay media and added to the plates containing the cellsand incubated together at 37° C. for 10 minutes. Additionally the assaywas also used to measure neutralization of IL-17A activity. Asub-maximal concentration (either EC₅₀ or EC₉₀, effective concentrationat 50 and 90 percent, respectively) of IL-17A was combined with serialdilutions of the human IL-17RA-Fc soluble receptor (ZGI), anti-humanIL-17A monoclonal antibody (R&D, MAB317), and anti-human IL-17Apolyclonal antibody (R&D, AF317NA) and incubated together at 37° C. for30 minutes in assay media prior to addition to cells. Followingpre-incubation, treatments were added to the plates containing the cellsand incubated together at 37° C. for 10 minutes.

Following incubation, cells were washed with ice-cold wash buffer andput on ice to stop the reaction according to manufacturer's instructions(BIO-PLEX Cell Lysis Kit, BIO-RAD Laboratories, Hercules, Calif.). 50μL/well lysis buffer was added to each well; lysates were pipetted upand down five times while on ice, then agitated on a microplate platformshaker for 20 minutes at 300 rpm and 4° C. Plates were centrifuged at4500 rpm at 4° C. for 20 minutes. Supernatants were collected andtransferred to a new micro titer plate for storage at −20° C.

Capture beads (BIO-ALEX Phospho-Iκβ-α Assay, BIO-RAD Laboratories) werecombined with 50 μL of 1:1 diluted lysates and added to a 96-well filterplate according to manufacture's instructions (BIO-PLEX PhosphoproteinDetection Kit, BIO-RAD Laboratories). The aluminum foil-covered platewas incubated overnight at room temperature, with shaking at 300 rpm.The plate was transferred to a microtiter vacuum apparatus and washedthree times with wash buffer. After addition of 25 μL/well detectionantibody, the foil-covered plate was incubated at room temperature for30 minutes with shaking at 300 rpm. The plate was filtered and washedthree times with wash buffer. Streptavidin-PE (50 μL/well) was added,and the foil-covered plate was incubated at room temperature for 15minutes with shaking at 300 rpm. The plate was filtered and washed twotimes with bead resuspension buffer. After the final wash, beads wereresuspended in 125 μL/well of bead suspension buffer, shaken for 30seconds, and read on an array reader (BIO-PLEX, BIO-RAD Laboratories)according to the manufacture's instructions. Data were analyzed usinganalytical software (BIO-PLEX MANAGER 3.0, BIO-RAD Laboratories).Increases in the level of the phosphorylated Iκβ-α transcription factorpresent in the lysates were indicative of an IL-17A receptor-ligandinteraction. For the neutralization assay, decreases in the level of thephosphorylated Iκβ-α transcription factor present in the lysates wereindicative of neutralization of the IL-17A receptor-ligand interaction.IC₅₀ (inhibitory concentration at 50 percent) values were calculatedusing GraphPad Prism® 4 software (GraphPad Software, Inc., San DiegoCalif.) and expressed as molar ratios for each reagent in theneutralization assay.

IL-17A induced Iκβ-α phosphorylation in a dose dependent manner with anEC₉₀ concentration determined to be 0.25 nM for both forms of theligand. IL-17A was neutralized by the anti-human IL-17A polyclonalantibody at a 1:1 molar ratio, by the human IL-17RA-Fc soluble receptorat a 7:1 molar ratio, and by the anti-human IL-17A monoclonal antibodyat a 50:1 molar ratio. TABLE 8 below shows IC50 values of representativeIL-17A neutralizing entities described herein, as compared to theIL-17RA-Fc and IL-17A polyclonal antibody controls. Results demonstratethat the IL-17A clones were effective at reducing the signals induced byhuman IL-17A.

TABLE 8 IC50 (nM) IL17A poly Ab 1.1 IL-17RA-Fc 2.7 cluster id IL-17A FabClone c87 (SQ7) M7.19 E7 5 c86 (SQ7) M7.19 D10 10 c97 (SQ7) M7.20 G6 21c95 (SQ7) M7.20 E5 79 c100 (SQ7) M7.24 G6 16 c99 (SQ7) M7.24 E8 56.0 c83(SQ7) M7.19 F4 27 c98 (SQ7) M7.24 E5 >133 c88 (SQ7) M7.20 F4 100 IL-17AscFv

Example 24 IL-23 Baf3/huIL-23R/huIL-12Rβ1 STAT3 Bioassay

The Baf3/KZ134/huIL-23R/huIL-12Rβ1 Clone 6 transfected cell line wasgenerated as described herein. Baf3/KZ134/huIL-23R/huIL-12RB1 Clone 6cells were washed two times with assay media (RPMI 1640 with L-Glutamineplus 10% fetal bovine serum, 1% Sodium Pyruvate, and 2 uMβ-Mercaptoethanol) before being plated out at 30,000 cells/well in96-well, round-bottom tissue culture plates. Serial dilutions ofrecombinant human IL-23 (ZGI CHO material or eBioscience's Insectheterodimer material) were made up in assay media and added to theplates containing the cells and incubated together at 37° C. for 15minutes. Additionally the assay was also used to measure neutralizationof IL-23 activity. A half maximal concentration (EC₅₀, effectiveconcentration at 50 percent) of IL-23 was combined with serial dilutionsof anti-human IL-12 p40 monoclonal antibody (Pharmingen), anti-humanIL-23 p19 polyclonal antibody (R&D, AF1716), and human IL-23R-Fc SolubleReceptor (ZGI) and incubated together at 37° C. for 30 minutes in assaymedia prior to addition to cells. Following pre-incubation, treatmentswere added to the plates containing the cells and incubated together at37° C. for 15 minutes.

Following incubation, cells were washed with ice-cold wash buffer andput on ice to stop the reaction according to manufacturer's instructions(BIO-FLEX Cell Lysis Kit, BIO-RAD Laboratories, Hercules, Calif.). Cellswere then spun down at 2000 rpm at 4° C. for 5 minutes prior to dumpingthe media. 50 μL/well lysis buffer was added to each well; lysates werepipetted up and down five times while on ice, then agitated on amicroplate platform shaker for 20 minutes at 300 rpm and 4° C. Plateswere centrifuged at 4500 rpm at 4° C. for 20 minutes. Supernatants werecollected and transferred to a new micro titer plate for storage at −20°C.

Capture beads (BIO-PLEX Phospho-STAT3 Assay, BIO-RAD Laboratories) werecombined with 50 μL of 1:1 diluted lysates and added to a 96-well filterplate according to manufacture's instructions (BIO-PLEX PhosphoproteinDetection Kit, BIO-RAD Laboratories). The aluminum foil-covered platewas incubated overnight at room temperature, with shaking at 300 rpm.The plate was transferred to a microtiter vacuum apparatus and washedthree times with wash buffer. After addition of 25 μL/well detectionantibody, the foil-covered plate was incubated at room temperature for30 minutes with shaking at 300 rpm. The plate was filtered and washedthree times with wash buffer. Streptavidin-PE (50 μL/well) was added,and the foil-covered plate was incubated at room temperature for 15minutes with shaking at 300 rpm. The plate was filtered and washed twotimes with bead resuspension buffer. After the final wash, beads wereresuspended in 125 μL/well of bead suspension buffer, shaken for 30seconds, and read on an array reader (BIO-PLEX, BIO-RAD Laboratories)according to the manufacture's instructions. Data were analyzed usinganalytical software (BIO-PLEX MANAGER 3.0, BIO-RAD Laboratories).Increases in the level of the phosphorylated STAT3 transcription factorpresent in the lysates were indicative of an IL-23 receptor-ligandinteraction. For the neutralization assay, decreases in the level of thephosphorylated STAT3 transcription factor present in the lysates wereindicative of neutralization of the IL-23 receptor-ligand interaction.IC₅₀ (inhibitory concentration at 50 percent) values were calculatedusing GraphPad Prism®4 software (GraphPad Software, Inc., San DiegoCalif.) and expressed as molar ratios for each reagent in theneutralization assay.

IL-23 induced STAT3 phosphorylation in a dose dependent manner with anEC₅₀ concentration determined to be 20 pM for ZGI A1806F and 30 pM foreBioscience heterodimer. Table 9 presents example IC50 data for theIL-23 positive controls (IL-23 p19 polyAb and IL-23R-Fc) and IL-23neutralizing entities described herein. These data indicate that theIL-23 neutralizing entities were efficacious and were equally or betterat reducing the effects of IL-23 as the controls.

TABLE 9 IC50 (nM) IL-23 p19 polyAb  65-126 IL-23R-Fc 11-14 cluster i.d.IL-23 Fab Clone c41 (SQ7) M7.12 B9 1.4 c29 (SQ7) M7.12 F9 2.3 c36 (SQ7)M7.9 G9 2.3 c29 (SQ7) M7.13 D7 7.4 c101 M7.3 D4 81 (SQ7) c27 (SQ7) M7.9A7 34.0 c87 (SQ7) M7.7 F5 >133 c103 M7.12 A7 >133 (SQ7) IL-23 scFv c305M7.36.B6 Range of (SQ22) 0.13-3.8 c304 M7.36.D3 31 (SQ22) c303M7.35.E9 >133 (SQ22) c302 M7.35.C9 >133 (SQ22)

Example 25 Cynomolgous Monkey IL-17A and IL-23p19 PolynucleotideSequences

CnIL-17A was cloned by PCR using a high fidelity thermostablepolymerase. (Expand, Roche Applied Science, Indianapolis, Ind.) Based onavailable sequence information from the UCSC Genome Browser for chimpand human IL-17A, oligonucleotides 56837 (SEQ ID NO: 27) and 56838 (SEQID NO: 28) were designed to amplify the cynomologus gene open readingframe. An oligo dT primer was used to initiate cDNA synthesis from totalRNA purified from PMA and Ionomycin stimulated cynomologus monkey PBMC'susing Superscripts Reverse Transcriptase following the manufacturersrecommendations (Invitrogen corp. Carlsbad, Calif.). PBMC's wereisolated from whole blood obtained from a 13 year old male M. fascularisanimal and stimulated for 4 hours in 10 ng/ml PMA and 0.5 ug/mlIonomycin. Total RNA isolated using RNeasy mini kit (Qiagen, Inc.Valencia, Calif.) following the manufacturers recommendations. Due tothe lack of sequence information for the cynomologus monkey, a numberPCR products were cloned into PCR4 TO (Invitrogen corp. Carlsbad,Calif.) for sequence comparisons. A nucleotide consensus sequence wasobtained from 12 clones and is shown in SEQ ID NO: 29.

CnIL-23p19 was cloned by PCR using a high fidelity thermostablepolymerase. (Expand, Roche Applied Science, Indianapolis, Ind.) Based onavailable sequence information from the UCSC Genome Browser for chimpand human IL-23p19, oligonucleotides 56846 (SEQ ID NO: 1016) and 56855(SEQ ID NO: 1017) were designed to amplify the cynomologus gene. Anoligo dT primer was used to initiate cDNA synthesis from total RNApurified from LPS, ploy IC, TNF and CD40 lig. stimulated monocytederived dendritic cell's using SuperscriptII Reverse Transcriptasefollowing the manufacturers recommendations (Invitrogen corp. Carlsbad,Calif.). PBMC's were isolated from whole blood obtained from a X yearold male M. fascularis animal and used to purify CD14+ cells using CD14non-human primate microbeads (Miltneyi Biotec, Bergisch Germany). CD14+cells were differentiated for 4 days with 10 ng/ml each hGMCSF and hIL-4then stimulated for 4 hours with 1 ug/ml LPS, 25 ng/ml polyIC, 20 ng/mlTNF and 10 ug/ml CD40 lig. Total RNA was isolated using RNeasy mini kit(Qiagen, Inc. Valencia, Calif.) following the manufacturersrecommendations. Due to the lack of sequence information for thecynomologus monkey, a number PCR products were cloned into PCR4 TO(Invitrogen corp. Carlsbad, Calif.) for sequence comparisons. Anucleotide consensus sequence was obtained from 16 clones and is shownin SEQ ID NO: 30.

Example 26 IL-12 Bioassay

Leukopheresis PBMC:

To obtain a consistent pool of PBMC's, normal human donors werevoluntarily apheresed. The leukopheresis PBMC were poured into a sterile500 ml plastic bottle, diluted to 400 ml with room temperature PBS+1 mMEDTA and transferred to 250 ml conical tubes. The 250 ml tubes werecentrifuged at 1500 rpm for 10 minutes to pellet the cells. The cellsupernatant was then removed and discarded. The cell pellets were thencombined and suspended in 400 ml PBS+1 mM EDTA. The cell suspension (25ml/tube) was overlaid onto Ficoll (20 ml/tube) in 50 ml conical tubes(total of 16 tubes). The tubes were centrifuged at 2000 rpm for 20minutes at room temperature. The interface layer (“buffy coat”)containing the white blood cells and residual platelets was collected,pooled and washed repeatedly with PBS+1 mM EDTA until the majority ofthe platelets had been removed. The white blood cells were thensuspended in 100 ml of ice-cold Cryopreservation medium (70% RPMI+20%FCS+10% DMSO) and distributed into sterile cryovials (1 ml cells/vial).The cryovials were placed in a −80° C. freezer for 24 hours beforetransfer to a liquid-nitrogen freezer. The white blood-cell yield from atypical apheresis is 0.5-1.0×10¹⁰ cells. Apheresis cells processed inthis manner contain T cells, B cells, NK cells, monocytes and dendriticcells.

Preparation of PHA Blasts:

T cells must be activated in order to express the IL-12 receptor and beable to respond to IL-12 and IL-23. Cryopreserved leukopheresis PBMCwere thawed, transferred to a sterile 50 ml conical tube, washed oncewith 50 ml of warm RPMI+10% heat-inactivated FBS+1 ug/ml DNAse I(Calbiochem), resuspended in 50 ml of fresh RPMI/FBS/DNAse medium andincubated in a 37° F. water bath for at least 1 hour to allow the cellsto recover from being thawed. The cells were then centrifuged and thecell-supernatant discarded. The cell pellet was resuspended in RPMI+10%FBS and distributed into sterile 75 cm² tissue culture flasks (1×10⁷cells/flask in 40 ml/flask). PHA-L (5 mg/ml stock in PBS) was added tothe cells at a final concentration of 5 ug/ml. The cells were thencultured at 37° C. in a humidified incubator for a total of 5 days. Thecells were “rested” for some experiments by harvesting the cells on theafternoon of day 4, replacing the culture medium with fresh RPMI+10% FBSwithout PHA-L (40 ml/flask) and returning the cells to their flasks andincubating at 37° C. the cells in a humidified incubator for theremainder of the 5 day culture period.

IL-12 and IL-23 Bioassays:

Three in vitro assays for detection of human IL-12 and IL-23 bioactivityon normal human T cells have been established: 1) IFN-gamma andMIP-1alpha production, 2) proliferation ([³H]-incorporation) and 3)STAT3 activation. Human PHA blasts (activated T cells) were harvested onday 5 of culture, suspended in fresh RPMI+10% FBS and plated at thedesired cell number per well in 96 well plates.

The inclusion of an IL-12 assay was used to determine specificity of theneutralizing entities described herein for IL-23p19 and not IL-12.

For the IFN-gamma production assay, the cells were plated at 1×10⁶/wellin flat-bottom 96-well plates. The cells were cultured at 37° C. in afinal volume of 200 ul/well with either medium alone, human IL-2 alone(10 ng/ml; R & D Systems), human IL-12 alone (graded doses; Invitrogen),human IL-23 alone (graded doses; ZGI lot #A1806; CHO-derived),anti-human CD28 mAb alone (graded doses; clone 28.2, e-Biosciences), oreach cytokine in combination with anti-human CD28 mAb. Triplicate wellswere set up for each culture condition. For the IFN-gamma productionassay, cell supernatants (120 ul/well) were harvested after 24-48 hoursof culturing the cells at 37° C. in a humidified incubator. HumanIFN-gamma and MIP-1alpha concentrations in these supernatants (pooledfor each triplicate) were measured using a commercial Luminex bead-basedELISA kit (Invitrogen) following the manufacturer's instructions.

Effects of IL-23 on IFN-gamma and MIP-1 alpha production were enhancedby culturing the cells with plate-immobilized anti-human CD3 mAb (5ug/ml) and soluble anti-human CD28 mAb (1 ug/ml) as well as harvestingthe supernatants (120 ul/well) after 48 hrs of culture at 37° C. thecells in a humidified incubator. Human IFN-gamma concentrations in thesesupernatants (pooled for each triplicate) were measured using acommercial Luminex bead-based ELISA kit (Invitrogen) following themanufacturer's instructions.

For the [³H]-incorporation assay the cells were plated at 2×10⁵cells/well in U-bottom 96-well plates. The cells were cultured at 37degrees C. for 72 hours. The cells were pulsed with 1 uCi/well of[³H]-Thymidine (Amersham) for the last 8 hours of this culture period.The cells were then harvested onto glass-fiber filters and the CPMs of[³H] incorporated were quantitated using a beta counter (Topcount NXT,Packard).

For each of these above endpoint parameters, effective neutralization ofactivity mediated by IL-23 was observed in the presence of anti-IL23p19neutralizing entities described herein at IC50 values that ranged from0.1 to ˜100 nM. There was no effect of the anti-IL-23p19 antagonists onneutralizing the effects mediated by IL-12, indicating specificity ofthe antagonists to IL-23p19.

STAT3 Bioassay:

For the STAT3 Bioassay the cells were plated at 2×10⁵ cells/well inU-bottom 96-well plates. Serial dilutions of human IL-12 (R&D) orrecombinant human IL-23 (ZGI CHO-derived material or eBioscience'sInsect heterodimer material) were prepared in assay media (RPMI 1640with L-Glutamine plus 10% fetal bovine serum), added to the platescontaining the cells and incubated together at 37° C. for 15 minutes.Additionally, the assay was also used to measure neutralization of IL-12and IL-23 activity using either commercially-available neutralizingreagents (as “controls”) or the anti-IL-23p19-containing neutralizingentities described herein. A half-maximal concentration (EC₅₀, effectiveconcentration at 50 percent) of IL-12 or IL-23 were combined with serialdilutions of anti-human IL-12 p40 monoclonal antibody (Pharmingen),anti-human IL-23 p19 polyclonal antibody (R&D, AF1716), human IL-23R-FcSoluble Receptor (ZGI), or any of the neutralizing entities describedherein, and incubated together at 37° C. for 30 minutes in assay mediaprior to addition to cells. Following pre-incubation, treatments wereadded to the plates containing the cells and incubated together at 37°C. for 15 minutes.

Following incubation, cells were washed with ice-cold wash buffer andput on ice to stop the reaction, according to manufacturer'sinstructions (BIO-PLEX Cell Lysis Kit, BIO-RAD Laboratories, Hercules,Calif.). Cells were then spun down at 2000 rpm at 4° C. for 5 minutesprior to removing the media. 50 ul/well lysis buffer was added to eachwell; lysates were pipetted up and down five times while on ice, thenagitated on a microplate platform shaker for 20 minutes at 300 rpm and4° C. Plates were centrifuged at 4500 rpm at 4° C. for 20 minutes.Supernatants were collected and transferred to a new micro titer platefor storage at −20° C.

Capture beads (BIO-PLEX Phospho-STAT3 Assay, MO-RAD Laboratories) werecombined with 50 ul of 1:1 diluted lysates and added to a 96-well filterplate according to manufacture's instructions (BIO-PLEX PhosphoproteinDetection Kit, BIO-RAD Laboratories). The aluminum foil-covered platewas incubated overnight at room temperature, with shaking at 300 rpm.The plate was transferred to a microtiter vacuum apparatus and washedthree times with wash buffer. After addition of 25 μL/well detectionantibody, the foil-covered plate was incubated at room temperature for30 minutes with shaking at 300 rpm. The plate was filtered and washedthree times with wash buffer. Streptavidin-PE (50 ul/well) was added,and the foil-covered plate was incubated at room temperature for 15minutes with shaking at 300 rpm. The plate was filtered and washed twotimes with bead resuspension buffer. After the final wash, beads wereresuspended in 125 ul/well of bead suspension buffer, shaken for 30seconds, and read on an array reader (BIO-PLEX, BIO-RAD Laboratories)according to the manufacture's instructions. Data were analyzed usinganalytical software (BIO-PLEX MANAGER 3.0, BIO-RAD Laboratories).

Increases in the level of the phosphorylated STAT3 transcription factorpresent in the lysates were indicative of an IL-12 or IL-23receptor-ligand interaction. For the neutralization assay, decreases inthe level of the phosphorylated STAT3 transcription factor present inthe lysates were indicative of neutralization of the IL-12 or IL-23receptor-ligand interaction. IC₅₀ (inhibitory concentration at 50percent) values were calculated using GraphPad Prism® 4 software(GraphPad Software, Inc., San Diego Calif.) and expressed as molarratios for each reagent and/or neutralizing entity in the neutralizationassay.

IL-12 and IL-23 both induced STAT3 phosphorylation in a dose dependentmanner with variation from donor to donor in PHA-activated human Tcells. EC50 values for IL-23 were in the range of 12-53 μM. IL-12 andIL-23 were both neutralized by the anti-human IL-12 p40 monoclonalantibody, whereas only IL-23 was neutralized by the anti-human IL-23 p19polyclonal antibody and human IL-23R-Fc controls, and by theanti-IL23p19 neutralizing entities described herein

For just one example, for one donor an EC₅₀ concentration was determinedto be 200 pM for IL-12 and 20 pM for IL-23 (using ZGI CHO-derivedprotein lot A1806F) and 30 pM for IL-23 (using the eBioscienceheterodimer). IL-12 and IL-23 were both neutralized by the commerciallyavailable anti-human IL-12 p40 monoclonal antibody. Only IL-23 wasneutralized by the commercially available anti-human IL-23 p19polyclonal antibody and human IL-23R-Fc soluble receptor (as“controls”), indicating that this assay is specific for its intendedcytokine activity/neutralization use. Results indicated that theefficacious neutralizing entities described herein were equally orbetter than the commercially available reagents at neutralizing theeffects of rhIL-23. (see Table 10 for representative exampleneutralizing values). Furthermore, results indicate that theneutralizing entities specifically inhibited rhIL-23 and not IL-12 (datanot shown; all data graphs indicated absolutely no neutralizing abilityof anti-IL-23p19 clones against IL-12 induced activity).

TABLE 10 IC50 Values for Neutralization of IL-23-Mediated pSTAT3Activity in Human PHA Blasts IC50 (nM) IL23 p19 pAb 52 IL-23R-Fc 3cluster i.d. IL-23 Fab Clone IMAC c41 (SQ7) M7.12 B9 1.8 c29 (SQ7) M7.12F9 5.3 c36 (SQ7) M7.9 G9 2.7 c29 (SQ7) M7.13 D7 7.9 c101 (SQ7) M7.3 D453 c27 (SQ7) M7.9 A7 85.0 c87 (SQ7) M7.7 F5 90-100 c103 (SQ7) M7.12 A790-100 c103 (SQ7) M7.13 A6 90-100

Example 27 Bioassay for Neutralization of huIL-17-Induced CytokineProduction in Human Small Airway Epithelial Cells (SAEC IL-12 PHABioassay)

Treatment of human small airway epithelial cells (SAEC) with rhIL-17induces the production of cytokines G-CSF, IL-6, and IL-8, which inturn, play a role in the pathology associated with the diseases forwhich an IL17/IL23p19 neutralizing entity would be efficacious. Theability of any of the neutralizing entities described herein to inhibitrhIL-17-mediated production of these cytokines is measured in thisbioassay, thus being predictive of in vivo efficacy against thesecytokines as well.

Method:

SAEC (cells and growth media purchased from Cambrex, Inc.) were platedat 8,000 cells/well in 96-well flat bottom tissue culture multi-wellplates, and placed in a 37° C., 5% CO₂ incubator. The following day,cells were treated with a dose range of the neutralizing entity incombination with 10-20 ng/mL rhIL-17. The ligand and neutralizing entitywere incubated together for 30 minutes at 37° C. before adding to thecells. Duplicate or triplicate wells were set up for each dose. After24-48 hours, supernatants are collected, and stored at −80° C. if notused directly. Before taking supernatants, wells were scanned byinverted microscope to make note of which wells had considerable celldeath. Those wells were not included in the final calculations.Supernatants were then assayed for cytokines huG-CSF, huIL-6, and huIL-8in a multiplex bead-based assay system (Bio-Rad Laboratories), and IC50determined.

Results:

In the presence of rhIL-17, the neutralizing entities described hereinwere efficacious at reducing cytokine production with IC50 valuesranging from 0.1-100 nM. Though there was some inter-experiment variabledue to different donors of SAEC, there were clearly some anti-IL17Aclones that had better neutralizing capacities than others. For example,with one donor SAEC, clone M7.19.E7 (SQ7_c87) had an IC50 of 31.1 nM toinhibit human IL-17A induced. G-CSF production, whereas with anotherdonor SAEC cultures, the same clone was able to inhibit IL-17A-mediatedG-CSF production with an IC50 of 13.4 nM. These data demonstrate thatalthough there was inter-donor/-experiment variability, the IL-17Aneutralizing entities were effective at neutralizing the biologicaleffects of IL-17A on human primary cells.

Example 28 Combination of Anti-IL-17 and Anti-IL-23p19 mAb in MurineColitis is More Efficacious than Either mAb Alone

IL-23 and IL-17 are important players in murine colitis and human IBD,via the actions of Th17 cells. IL-23 and IL-17 are upregulated incolitis and IBD, and neutralization of either cytokine alone isefficacious in several animal models of colitis (Fujino et al, Gut,2003, 52:65-70; Schmidt et al, Inflamm Bowel Dis. 2005, 11:16-23; Yen etal, J Clin Invest. 2006, 116:1310-1316; Zhang et al, Inflamm Bowel Dis.2006, 12:382-388; Kullberg et al, J Exp Med. 2006, 203:2485-94). SinceIL-23 is important for the maintenance, differentiation, and/orinduction of Th17 cells, neutralization of both cytokines would be moreefficacious at reducing disease than either cytokine alone. The currentexample provides data to support this in oxazalone-induced colitismodel, which resembles human ulcerative colitis (UC).

Methods:

For this experiment, 40 C57BL/10 female mice (obtained from Harlan) wereused. On day −5, mice were treated topically with 200 ul of 3.0% (w/v)oxazalone in 100% ethanol (“sensitization”) on the abdomen. On day 0,all mice receive intrarectal injections (120 uL each) of 2.0% (w/v)oxazalone in 50% ethanol while under light isoflurane gas anesthesia(“challenge”). Mice were monitored for disease using a Disease ActivityIndex (DAI) score, which includes stool consistency, body weight, andblood in stool. For mAb treatments, mice were administered one of thefollowing, via i.p. injection on days −5, −3, and −1: PBS, 50 ugneutralizing anti-mouse IL-17, 50 ug neutralizing anti-mouse IL-23p19mAb, or a combination of the anti-IL17+IL-23p19 mAb's.

Mice were euthanized on day 2. Serum was collected and stored for lateranalysis; colons were removed and observed for any gross sips of colitis(lesion, colon shortening, and colon wall thickening). Colons were thencut longitudinally and processed for histology and for 24 h coloncultures.

Results:

There was a significant reduction (p=0.0216; 54% reduction compared toPBS group) in DAI score and significant (p<0.05) improvement inhistological morphology (e.g. reduced colonic damage and reducedinflammation) in mice treated with the combination ofanti-IL-17+anti-IL-23p19 antibodies, compared to PBS and either mAbalone. Consistent with these benefits, mice treated with the combinationof antibodies also had significant improvements (p<0.0112) in colonshortening compared to PBS-treated mice and mice treated with either ofthe antibodies alone. In the production of inflammatory cyokines fromcolon cultures, oxazolone mice treated with the anti-IL-17+IL-23p19combination antibodies had less increases in IL-1b, IL-12, IL-13, IL-17,IL-23, and TNF-a compared to PBS-treated oxazalone mice. Treatment withthe combination of anti-IL-17+IL-23p19 antibodies also resulted insignificant reductions in serum IL-12, IL-17, IL-23, and TNF-aconcentrations compared to PBS-treated mice.

Therefore, the delivery of the therapeutic combination of anti-IL-17 andanti-IL-23p19 antibodies was significantly more efficacious in reducingcolitis from several aspects: disease symptoms, pathology, andinflammatory cytokine production. Taken together, these results indicatethat the therapeutic combination of IL-17 and IL-23/p19 neutralizingantibodies is more efficacious in the treatment of oxazalone colitis asa model of human IBD. The therapeutic combination can reduce clinicaldisease symptoms and works at the molecular level to reduceinflammation, tissue damage, inflammatory cytokines, and othermechanisms known to be affected in this manner.

Example 29 Measurement of Binding Affinities of Anti-IL-17A Molecules toIL-17A Via Surface Plasmon Resonance (Biacore)

Anti-IL-17A entities described herein were evaluated for their bindingaffinities to IL-17A using surface plasmon resonance and Biacore T-100instrument (GE Healthcare). Recombinant human IL-17A was immobilized tothe sensor chip, followed by passing the antagonists over theimmobilized ligand to attain affinity measurements. These methods allowfor binding affinity measurements of the IL-17A neutralizing entitiesfor their respective ligand (IL-17A) and also demonstrate specificityfor IL-17A by displaying no binding to similar but different ligands,such as other members of the IL-17 family.

To determine the best conditions for immobilization, a series of pHscouting experiments were performed. For these experiments, recombinanthuman IL-17A (ZGI lot A1781F) was diluted to 100 nM in five differentimmobilization buffers; Acetate-4.0, Acetate-4.5, Acetate-5.0,Acetate-5.5, and Borate-8.5. Using Immobilization Scouting Wizard, theseconditions were tested and at the end Acetate-4.0 was the best conditionfor immobilization.

For the immobilization procedure, IL-17A protein was diluted to 10 ug/mlin Acetate-4.5, and then immobilized onto a Series S Sensor Chip (CM5,GE Healthcare/Biacore #BR-1006-68) using the amine coupling kit andBiacore Immobilization Wizard. Briefly, the level of immobilization wastargeted to 300 Biacore Resonance Units (RU), and IL-17A was onlyinjected over an active flow cell. After the immobilization procedure,active sites on the flow cell were blocked with ethanolamine.Non-specifically bound protein was removed by washing with 50 mM NaOH.The final immobilization level was 466 RU. The reference cell wasactivated and then blocked with ethanolamine.

For the kinetic run, serial dilutions of the control proteins,IL-17RA-Fc (ZGI lot A1763F) or IL-17A monoclonal antibody (R&D MAB317),or anti-IL-17A neutralizing entities described herein, were prepared in1×FIBS-EP+ buffer. Duplicate injections of each concentration wereperformed. The analyte injections were at 30 ul/min with a dissociationtime of 600 seconds. Buffer injections were also performed to allow forsubtraction of instrument noise and drift.

Regeneration buffers supplied in the Regeneration Scouting Kit (GEHealthcare/Biacore #BR-1005-56) were used to determine regenerationconditions. Standard Biacore methods were performed to define the bestregeneration conditions for an IL-17A surface using IL-17RA-Fc at 100nM. Mildest conditions were tested first and moved up in strength. Thefinal regeneration condition chosen was a 1-60 sec pulse of 10 mM H3PO4followed by a 1-60 sec pulse of 1×HSB-EP buffer at 50 ul/min.

As a check for IL-17A specific interactions, similar procedures werefollowed using immobilized recombinant human IL-17F (ZGI lot A 275F).

Data were analyzed using Biacore Evaluation software (v.1.1.1) to definethe kinetic values of the interaction of IL-17A with the controlproteins (IL-17A monoclonal antibody and IL-17RA-Fc) and IL-17Aneutralizing entities described herein. Baseline stability was assessedto ensure that the regeneration step provided a consistent bindingsurface throughout the sequence of injections. Binding curves werenormalized by double-referencing, and duplicate injection curves werechecked for reproducibility. The resulting binding curves were globallyfit to the bivalent interaction model.

The data demonstrate high affinity binding of human IL-17A to thecontrol antagonist proteins (IL-17RA-Fc and anti-IL-17A monoclonalantibody) and to the IL-17A neutralizing entities described herein.There was no binding of the control IL-17A antagonist proteins or theanti-IL-17A neutralizing entities to IL-17F, thus providing evidence forspecificity of targeting IL-17A.

Specifically, human IL-17A demonstrates dissociation equilibriumconstants (KD) for IL-17R-Fc to be approximately 5 nM and approximately2 nM for the II 17A monoclonal antibody. Neutralizing entities describedherein display a large range of affinities, but are well within thisrange (0.4-<15 nM), thus demonstrating comparable binding affinities.

Example 30 Off-Rate Analysis of Anti-IL-17A Molecules to IL-17A ViaSurface Plasmon Resonance (Biacore)

Anti-IL-17A entities described herein were evaluated for bindingoff-rates to IL-17A using surface plasmon resonance and Biacore T-100instrument (GE Healthcare). Off-rate analysis is thought to helpestimate the interaction that occurs in vivo, since a slow off-ratewould predict a greater degree of interaction over long period of time.For these experiments, recombinant human IL-17A was immobilized to thesensor chip, followed by passing the antagonists over the immobilizedligand to attain off-rate analyses. Similar to the Biacore affinityexample (see EXAMPLE 29), IL-17F was used as the negative control, inorder to demonstrate IL-17A specificity.

To determine the best conditions for immobilization, a series of pHscouting experiments were performed. For these experiments, recombinanthuman. IL-17A (ZGI lot A1781F) or human IL-17F (ZGI lot A1275F) wasdiluted to 100 nM in five different immobilization buffers; Acetate-4.0,Acetate-4.5, Acetate-5.0, Acetate-5.5, and Borate-8.5. UsingImmobilization Scouting Wizard, these conditions were tested and at theend, Acetate-4.0 was the best condition for immobilization.

For the immobilization procedure, human IL-17A (native and biotinylated)and IL-17F were diluted to 10 ug/ml in Acetate-4.0. All three proteinswere immobilized onto a Series S Sensor Chip (CM5, GE Healthcare/Biacore#BR-1006-68) using the amine coupling kit and Biacore ImmobilizationWizard. Briefly, the level of immobilization was targeted to 500 BiacoreResonance Units (RU). Flow cell 1 was used as the reference, andtherefore was only activated and then blocked with ethanolamine. Thenative form of IL-17A was immobilized to flow cell 2, biotinylatedIL-17A was immobilized to flow cell 3 and IL-17F was immobilized to flowcell 4. Analyte was injected only over active flow cells (flow cells 2,3 and 4). After the immobilization, the active sites on the flow cellwere blocked with ethanolamine. Non-specifically bound protein wasremoved by washing with 50 mM. NaOH. The final immobilization level forflow cell 1 (reference) was 175.0 RU and fell between 669.5 and 695.6 RUfor the active flow cells (flow cells 2-4).

Samples of the anti-IL-17A neutralizing entities were diluted to 100 nMin 1×HBS-EP+ buffer (GE Healthcare/Biacore, #BR-1006-69). IL-17RA-Fc andIL-17A monoclonal antibody were also prepared at 100 nM in 1×HBS-EP+buffer as positive controls. Duplicate injections of each sample wereperformed. The analyte injections were at 30 ul/min with a dissociationtime of 600 seconds. Buffer injections were also performed to allow forsubtraction of instrument noise and drift.

Regeneration buffers supplied in a regeneration scouting kit were usedto determine regeneration conditions. Using this procedure, the optimalregeneration condition was found to be 60 seconds of 10 mM H3PO4followed with 60 seconds of 1×HBS-EP+ buffer injections at a flow rateof 50 ul/minute.

Data were evaluated using Biacore Evaluation software to define theoff-rate of the interactions of anti-IL-17A neutralizing entities toimmobilized IL-17A. Baseline stability was assessed to ensure that theregeneration step provided a consistent binding surface throughout thesequence of injections. Binding curves were normalized bydouble-referencing. Duplicate injection curves were checked forreproducibility and the resulting binding curves were globally-fit tothe bivalent interaction model.

All data for Biacore kinetics experiments were collected and analyzedusing Biacore Evaluation Software v1.1.1. Final sensorgrams of theIL-17A neutralizing entities were all y-normalized immediately beforedissociation phase. These dissociation curves were used to rank samplesaccording to their off-rates, slowest to fastest. Off-rates werecompared to the positive controls, IL-17RA-Fc (off-rate of 2.53×10-5sec-1).

TABLE 11a anti-IL-17A Fabs Off-rat Rankings: Panel #1 Sample (clone andcluster id) off-rate (slow to fast) IL17RA k_(d) = 2.53E−05 M7.20.G6(c97) slow k_(d) M7.19.E7 (c87) | M7.19.F4 (c83) | M7.24.E8 (c99) |M7.20.E5 (c95) | M7.19.D10 (c86) ↓ M7.24.G6 (c100) fast k_(d)***M7.24.E5 (c98)

TABLE 11b anti-IL-17A Fabs Off-rate Rankings: Panel #2 Sample off-rate(slow to fast) IL17RA k_(d) = 2.53E−05 M7.19.E7 (c87) slow k_(d)M7.24.A5 (c88) | M7.24.A4 (c94) | M7.20.F11 (c96) | M7.20.A9 (c90) ↓M7.20.C10 (c94) fast k_(d) M7.24.D8

TABLE 11c anti-IL-17A scFv Off-rate Rankings RANKING Clone # 1 M7.42_C111 M7.42_C09 2 M7.46_A02 2 M7.44_F09 3 M7.73_B05 3 M7.44_F09 3 M7.41_A024 M7.73_D03 4 M7.76_D11 4 M7.70_G02 4 M7.70_G08 4 M7.68_F06 4 M7.72_B105 M7.70_F05 5 M7.46_B08 6 M7.46_E07 7 M7.68_E02 7 M7.75_B06 7 M7.70_E107 M7.44_H04 7 M7.42_A10 7 M7.46_D08 7 M7.42_E10 7 M7.44_E05 7 M7.70_G117 M7.45_H08 7 M7.69_C07 7 M7.70_B04 7 M7.70_D07 7 M7.68_G06 7 M7.73_G047 M7.74_C11 7 M7.70_H11 7 M7.73_F04 7 M7.72_F03 7 M7.46_B05 7 M7.72_C118 M7.72_E03

Example 31 Measurement of Binding Affinities of Anti-IL-23p19 Moleculesto IL-23 Via Surface Plasmon Resonance (Biacore)

Anti-IL-23p19 entities described herein were evaluated for their bindingaffinities to IL-23 using surface plasmon resonance and Biacore T-100instrument (GE Healthcare). Recombinant human IL-23 was immobilized tothe sensor chip, followed by passing the antagonists over theimmobilized ligand to attain affinity measurements. These methods allowfor binding affinity measurements of the IL-23p19 neutralizing entitiesfor their respective ligand (IL-23) and also demonstrate specificity forIL-23 by displaying no binding to similar but different ligands, such asIL-12.

To determine the best conditions for immobilization, a series of pHscouting experiments were performed. For these experiments, recombinanthuman IL-23 (ZGI lot A1806F) was diluted to 100 nM in five differentimmobilization buffers; Acetate-4.0, Acetate-4.5, Acetate-5.0,Acetate-5.5, and Borate-8.5. Using Immobilization Scouting Wizard, theseconditions were tested and at the end Acetate-4.5 was the best conditionfor immobilization.

For the immobilization procedure, IL-23 protein was diluted to 10 ug/mlin Acetate-4.5, and then immobilized onto a Series S Sensor Chip (CM5,GE Healthcare/Biacore #BR-1006-68) using the amine coupling kit andBiacore Immobilization Wizard. Briefly, the level of immobilization wastargeted to 300 Biacore Resonance Units (RU), and IL-23 was onlyinjected over an active flow cell. After the immobilization procedure,active sites on the flow cell were blocked with ethanolamine.Non-specifically bound protein was removed by washing with 50 mM NaOH.The final immobilization level was 466 RU. The reference cell wasactivated and then blocked with ethanolamine.

To determine the optimal contact time, RL (resonance signal of theligand) testing was performed. Control proteins, soluble IL-23R-Fc (ZGIlot A1913F) and an anti-IL-23p19 polyclonal antibody (R&D Systems), werediluted to 100 nM in 1×HSB-EP+ running buffer (GE Healthcare/Biacore,#BR-1006-69). The IL-23R-Fc or IL-23p19 polyclonal antibody wereinjected over both the active and reference cells for three differentcontact times. From this RL testing, the contact time for the IL-23p19polyclonal antibody was determined to be 60 seconds and for IL-23R-Fc,it was 300 seconds.

For the kinetic run, serial dilutions of the control proteins, IL-23R-Fcor IL-23p19 polyclonal antibody, or anti-IL-23p19 neutralizing entitiesdescribed herein, were prepared in 1×HBS-EP+ buffer. Duplicateinjections of each concentration were performed. The analyte injectionswere at 30 ul/min with a dissociation time of 600 seconds. Bufferinjections were also performed to allow for subtraction of instrumentnoise and drift.

Regeneration buffers supplied in the Regeneration Scouting Kit (GEHealthcare/Biacore #BR-1005-56) were used to determine regenerationconditions. This run was performed manually, starting from the leastmild condition. At the end of this procedure, the optimal regenerationcondition was 60 seconds of 2M Magnesium Chloride followed with 60seconds of 1×HBS-EP+ buffer injections at a flow rate of 50 ul/min.

As a check for IL-23 specific interactions, similar procedures werefollowed using immobilized recombinant human IL-12 (R&D Systems).

Data were analyzed using Biacore Evaluation software (v.1.1.1) to definethe kinetic values of the interaction of IL-23 with the control proteins(IL-23p19 polyclonal antibody and IL-23R-Fc) and IL-23p19 neutralizingentities described herein. Baseline stability was assessed to ensurethat the regeneration step provided a consistent binding surfacethroughout the sequence of injections. Binding curves were normalized bydouble-referencing, and duplicate injection curves were checked forreproducibility. The resulting binding curves were globally fit to thebivalent interaction model.

The data demonstrated high affinity binding of human IL-23 to thecontrol antagonist proteins, IL-23R-Fc and anti-IL-23p19 polyclonalantibody, with affinity constants (KD) of 0.8 nM and 5.1 nM,respectively. The IL-23p19 neutralizing entities described herein werewithin this range. For example, clone M7.36_B06 (SQ22_c305) as an scFvhad a KD (M) of 3.4×10⁻⁸. There was no binding of the control IL-23antagonist proteins or the anti-IL-23p19 neutralizing entities to IL-12,thus providing evidence for specificity of targeting IL-23.

Example 32 Off-Rate Analysis of Anti-IL-23p19 Molecules to IL-23 ViaSurface Plasmon Resonance (Biacore)

Anti-IL-23p19 entities described herein were evaluated for bindingoff-rates to IL-23 using surface plasmon resonance and Biacore T-100instrument (GE Healthcare). Off-rate analysis is thought to helpestimate the interaction that occurs in vivo, since a slow off-ratewould predict a greater degree of interaction over long period of time.For these experiments, recombinant human IL-23 was immobilized to thesensor chip, followed by passing the antagonists over the immobilizedligand to attain off-rate analyses. Similar to the Biacore affinityexample (see EXAMPLE 31), IL-12 was used as the negative control, inorder to demonstrate IL-23 specificity.

To determine the best conditions for immobilization, a series of pHscouting experiments were performed. For these experiments, recombinanthuman IL-23 (ZGI lot A1806F) was diluted to 100 nM in five differentimmobilization buffers; Acetate-4.0, Acetate-4.5, Acetate-5.0,Acetate-5.5, and Borate-8.5. Using immobilization scouting wizard, theseconditions were tested, and at the end Acetate-4.5 was the bestcondition for immobilization.

For the immobilization procedure, human IL-23 (native and biotinylated)and IL-12 were diluted to 10 ug/ml in Acetate-4.5. All three proteinswere immobilized onto a Series S Sensor Chip (CM5, GE Healthcare/Biacore#BR-1006-68) using the amine coupling kit and Biacore ImmobilizationWizard. Briefly, the level of immobilization was targeted to 300 BiacoreResonance Units (RU). Flow cell 1 was used as the reference, andtherefore was only activated and then blocked with ethanolamine. Thenative form of IL-23 was immobilized to flow cell 2, biotinylated IL-23was immobilized to flow cell 3 and IL-12 was immobilized to flow cell 4.Analyte was injected only over active flow cells (flow cells 2, 3 and4). After the immobilization, the active sites on the flow cell wereblocked with ethanolamine. Non-specifically bound protein was removed bywashing with 50 mM NaOH. The final immobilization levels fell between379 and 415 RU.

Samples of the anti-IL-23p19 neutralizing entities were diluted to 100nM in 1×HBS-EP+ buffer (GE Healthcare/Biacore, #BR-1006-69). IL-23R-Fcwas also prepared at 100 nM in 1×HBS-EP+ buffer as a positive control.Duplicate injections of each sample were performed. The analyteinjections were at 30 uL/min with dissociation time of 600 seconds.Buffer injections were also performed to allow for subtraction ofinstrument noise and drift.

Regeneration buffers supplied in a regeneration scouting kit were usedto determine regeneration conditions (RR-2007001). Using this procedure,the optimal regeneration condition was found to be 60 seconds of 2MMagnesium Chloride followed with 60 seconds of 1×HBS-EP+ bufferinjections at a flow rate of 50 ul/min.

Data were evaluated using Biacore Evaluation software to define theoff-rate of the interactions of anti-IL-23p19 neutralizing entities toimmobilized IL-23. Baseline stability was assessed to ensure that theregeneration step provided a consistent binding surface throughout thesequence of injections. Binding curves were normalized bydouble-referencing. Duplicate injection curves were checked forreproducibility and the resulting binding curves were globally-fit tothe bivalent interaction model.

TABLE 12a anti-IL-23p19 Fab off-rate rankings Sample (cluster id)off-rate (slow to fast) IL23 Receptor k_(d) = 4E−4 M7.7.F5 (c87) slowk_(d) M7.12.F9 (c29) M7.12.A7 (c103) M7.13.A6 (c103) M7.9.G9 (c36)medium k_(d) M7.12.B9 (c41) M7.9.A7 (c27) fast k_(d) M7.3.D4 (c101)

TABLE 12b anti-IL-23p19 off-rate rankings: Panel #1 Sample (cluster id)off-rate (slow to fast) IL23 Receptor k_(d) = 4E−4 M7.36.B6 (c305) slowM7.35.E9 (c303) | M7.36.D3 (c304) ↓ M7.35.C9 (c302) fast

TABLE 12c anti-IL-23p19 scFv Off-rate Rankings Ranking Clone # 1M7.36_C06 1 M7.50_C03 2 M7.36_B03 2 M7.66_A07 2 M7.66_G05 3 M7.36_A07 3M7.66_E06 3 M7.67_A03 3 M7.67_F12 3 M7.50_D05 3 M7.65_F07 4 M7.66_D07 5M7.67_B07 5 M7.67_G11 5 M7.64_F07 5 M7.50_D07 5 M7.65_F02 5 M7.49_A07 5M7.67_F11 6 M7.67_C09 7 M7.66_A08 7 M7.67_G09 7 M7.66_D08 7 M7.49_E11 7M7.67_F10 8 M7.50_D03 9 M7.65_E09

Example 33 Characterization of Anti-IL-17A Fabs

Anti-IL-17A Fabs from 16 different E. coli clones demonstrated theability to neutralize the activity of IL-17A in a cell-basedneutralization assays. The functional binding properties of theseanti-IL-17A neutralizing Fabs were additionally characterized usingcompetitive binding (epitope binning) experiments.

Competitive Epitope Binding (Epitope Binning)

Epitope binning experiments were performed to determine whichanti-IL-17A Fabs are capable of binding simultaneously to human IL-17A.Anti-IL-17A Fabs that compete for the same, or an overlapping, bindingsite (epitope) on the antigen are not able to bind simultaneously andare functionally grouped into a single family or “epitope bin”.Anti-IL-17A Fabs that do not compete for the same binding site on theantigen are able to bind simultaneously and are grouped into separatefamilies or “epitope bins”. Experiments were performed using a Biacore3000™ instrument. Biacore is only one of a variety of assay formats thatare routinely used to assign panels of antibody fragments and monoclonalantibodies to epitope bins. Many references (e.g. The Epitope MappingProtocols, Methods in Molecular Biology, Volume 6,6 Glenn E. Morris ed.)describe alternative methods that can be used (by those skilled in theart) to “bin” the antibody fragments, and would be expected to providecomparable data regarding the binding characteristics of the anti-IL-17AFabs to human IL-17A. Epitope binning experiments were performed withsoluble, native antigen.

Materials and Methods

Epitope binning studies were performed on a Biacore3000™ system(Biacore, Uppsalla Sweden). Methods were programmed using BiacoreControl Software, v 3.2. Human IL-17A (ZymoGenetics) was covalentlyimmobilized to a Biacore CM5 sensor chip. Subsequently, the firstanti-IL-17A Fab (primary) of a test pair was injected and allowed tospecifically bind to the antigen immobilized on the sensor chip. TheBiacore instrument measures the mass of protein bound to the sensor chipsurface, and thus, immobilization of the antigen and specific binding ofthe primary Fab of a test pair were verified for each test cycle. Carewas taken to confirm that all the binding sites for the primary Fab weresaturated prior to exposure to the secondary anti-IL-17A Fab of the testpair. Following the binding of the primary Fab of the test pair, asecondary anti-IL-17A Fab was injected and allowed to bind to theimmobilized antigen. If the secondary anti-IL-17A Fab was capable ofbinding the antigen simultaneously with the primary Fab, an increase inmass on the surface of the chip, or binding, was detected. If, however,the secondary anti-IL-17A Fab was not capable of binding the antigensimultaneously with the primary Fab, no additional mass, or binding, wasdetected. Each anti-IL-17A Fab tested against itself was used as thenegative control to establish the level of the background (no-binding)signal.

A series of experiments were completed to test the binding properties ofpurified anti-IL-17A Fabs from 15 clones (M7.19 E7; M7.24 E5; M7.20 G6;M7.24 E8; M7.19 F4; M7.24 G6; M7.19 D10; M7.20 E5; M7.24 A5; M7.20 C10;M7.20 F11; M7.20 A9; M7.24 C2; M7.20 F4; M7.24 D8) generated againsthuman IL-17A. IL-17A was covalently immobilized using EDC:NHS to adensity of approximately 2000 RU. Previous experiments have demonstratedthat this type of immobilization does not interfere with the binding ofthe anti-IL-17A Fabs. Each anti-IL-17A Fab was tested as the primaryanti-IL-17A Fab in combination with a subset of secondary anti-IL-17AFabs. The primary anti-IL-17A Fabs were tested at a concentration of 200nM and a flow rate of 10 μL/min, while the secondary anti-IL-17A Fabswere tested at a concentration of 100 nM and a flow rate of 50 μL/min.Experiments were performed at 25° C. Between cycles, the antigen on thechip was regenerated with 10 mM phosphoric acid. Data was compiled usingBioEvaluation 4.1 RCI software, then loaded into Excel™ for additionaldata processing.

Results:

A subset of the purified anti-IL-17A Fabs that bind human IL-17A werecharacterized and assigned into epitope bins. The signal (RU, responseunits) reported by the Biacore is directly correlated to the mass on thesensor chip surface. Once the level of background signal (RU) associatedwith the negative controls was established (a single anti-IL-17A Fabused as both the primary and secondary Fabs), the binning results werereported as either positive or negative binding. Positive bindingindicates that two different anti-IL-17A Fabs are capable of binding theantigen simultaneously. Negative binding indicates that two differentanti-IL-17A Fabs are not capable of binding the antigen simultaneously.The differential between positive and negative response values in thisexperiment was significant and allowed for an unambiguous assignment ofthirteen of the anti-IL-17A Fabs into two distinct families or epitopebins. Two binding epitopes were identified within this set ofneutralizing anti-IL-17A Fabs, and multiple E. coli clones were found tobind to one of the epitopes. The first epitope bin was comprised ofanti-IL-17A Fabs from clones M7.19 E7; M7.20 G6; M7.24 E8; M7.19 F4;M7.19 D10; M7.20 E5; M7.24 A5; M7.20 C10; M7.20 F11; M7.20 A9; M7.24 C2;M7.20 F4. The second bin was comprised of the anti-IL-17A Fab from cloneM7.24 G6.

Example 34 Characterization of Anti-IL-23p19 Fabs

Anti-IL-23p19 Fabs from 9 different E. coli clones demonstrated theability to neutralize the activity of IL-23 in a cell-basedneutralization assays. The functional binding properties of theseanti-IL-23p19 neutralizing Fabs were additionally characterized usingcompetitive binding (epitope binning) experiments.

Competitive Epitope Binding (Epitope Binning)

Epitope binning experiments were performed to determine whichanti-IL-23p19 Fabs are capable of binding simultaneously to human IL-23.Anti-IL-23p19 Fabs that compete for the same, or an overlapping, bindingsite (epitope) on the antigen are not able to bind simultaneously andare functionally grouped into a single family or “epitope bin”.Anti-IL-23p19 Fabs that do not compete for the same binding site on theantigen are able to bind simultaneously and are grouped into separatefamilies or “epitope bins”. Experiments were performed using a Biacore3000™ instrument. Biacore is only one of a variety of assay formats thatare routinely used to assign panels of antibody fragments and monoclonalantibodies to epitope bins. Many references (e.g. The Epitope MappingProtocols, Methods in Molecular Biology, Volume 6,6 Glenn E. Morris ed.)describe alternative methods that can be used (by those skilled in theart) to “bin” the antibody fragments, and would be expected to providecomparable data regarding the binding characteristics of theanti-IL-23p19 Fabs to human IL-23. Epitope binning experiments wereperformed with soluble, native antigen.

Materials and Methods

Epitope binning studies were performed on a Biacore3000™ system(Biacore, Uppsalla Sweden). Methods were programmed using BiacoreControl Software, v 3.2. Human IL-23 (ZymoGenetics) was covalentlyimmobilized to a Biacore CM5 sensor chip. Subsequently, the firstanti-IL-23p19 Fab (primary) of a test pair was injected and allowed tospecifically bind to the antigen immobilized on the sensor chip. TheBiacore instrument measures the mass of protein bound to the sensor chipsurface, and thus, immobilization of the antigen and specific binding ofthe primary Fab of a test pair were verified for each test cycle. Carewas taken to confirm that all the binding sites for the primary Fab weresaturated prior to exposure to the secondary anti-IL-23p19 Fab of thetest pair. Following the binding of the primary Fab of the test pair, asecondary anti-IL-23p19 Fab was injected and allowed to bind to theimmobilized antigen. If the secondary anti-IL-23p19 Fab was capable ofbinding the antigen simultaneously with the primary Fab, an increase inmass on the surface of the chip, or binding, was detected. If, however,the secondary anti-IL-23p19 Fab was not capable of binding the antigensimultaneously with the primary Fab, no additional mass, or binding, wasdetected. Each anti-IL-23p19 Fab tested against itself was used as thenegative control to establish the level of the background (no-binding)signal.

A single experiment was completed to test the binding properties ofpurified anti-IL-23p19 Fabs from 9 clones (M7.3 D4; M7.7 F5; M7.9 A7;M7.9 G9; M7.12 A7; M7.12 B9; M7.12 F9; M7.13 A6; M7.13 D7) generatedagainst human IL-23. IL-23 was covalently immobilized using EDC:NHS to adensity of approximately 2000 RU. Previous experiments have demonstratedthat this type of immobilization does not interfere with the binding ofthe anti-IL-23p19 Fabs. Each anti-IL-23p19 Fab was tested as the primaryFab in combination with the entire panel of secondary anti-IL-23p19Fabs. The primary anti-IL-23p19 Fabs were tested at a concentration of210 nM and a flow rate of 10 μL/min, while the secondary anti-IL-23p19Fabs were tested at a concentration of 60 nM and a flow rate of 50μL/min. Experiments were performed at 25° C. Between cycles, the antigenon the chip was regenerated with 2 M MgCl2. Data was compiled usingBioEvaluation 4.1 RCI software, then loaded into Excel™ for additionaldata processing.

Results:

A subset of the purified anti-IL-23p19 Fabs that bind human IL-23 werecharacterized and assigned into epitope bins. The signal (RU, responseunits) reported by the Biacore is directly correlated to the mass on thesensor chip surface. Once the level of background signal (RU) associatedwith the negative controls was established (a single anti-IL-23p19 Fabused as both the primary and secondary Fabs), the binning results werereported as either positive or negative binding. Positive bindingindicates that two different anti-IL-23p19 Fabs are capable of bindingthe antigen simultaneously. Negative binding indicates that twodifferent anti-IL-23p19 Fabs are not capable of binding the antigensimultaneously. The differential between positive and negative responsevalues in this experiment was significant and allowed for an unambiguousassignment of six of the anti-IL-23p19 Fabs into two distinct familiesor epitope bins. Two binding epitopes were identified within this set ofneutralizing anti-IL-23p19 Fabs, and multiple E. coli clones were foundto bind to each epitope. The first epitope bin was comprised ofanti-IL-23p19 Fabs from clones M7.12 139; M7.9 G9; and M7.13 D7. Thesecond bin was comprised of anti-IL-23p19 Fabs from clone M7.7 F5; M7.12A7; and M7.13 A6.

Example 35 Determination of Ability of Anti-Human IL-17A NeutralizingEntities to Cross-React and Neutralize Cynomolgous Monkey IL-17A-InducedActivity in IL-17A Bioassays

Species cross-reactivity studies (especially for non-human primatecross-reactivity) are an important activity to demonstrate fortherapeutic antagonist development strategies. In order to determinewhether the anti-human IL-17A neutralizing entities described herein maycross-react and neutralize the activity induced by cynomolgous IL-17A(and therefore, justify the cynomolgous monkey as a viable testspecies), it was first necessary to demonstrate comparable activitiesinduced by both recombinant human and cynomolgous monkey IL-17A in thebioassays employed here for testing neutralization. The methods forIL-17A activity assays described in EXAMPLES 5, 23 and 27 were used witha full range (0-112 nM) of either recombinant human IL-17A (ZGI lotA1781F) or recombinant cynomolgous IL-17A (ZGI lot A1906F). Resultsindicate that the activity induced by the human and cynomolgous IL-17Aproteins used here were nearly identical in all three assays, yieldingindistinguishable curves and EC50 values (identical between species foreach experiments, and ranged from 0.22-0.41 nM amongst independentreplicate experiments performed on different days).

Inclusion of the human IL-17RA-Fc was able to neutralize the effects ofeither human IL-17A or cynomolgous IL-17A with IC50 values nearlyidentical for either species (i.e. 2.9 nM for neutralization againsthuman IL-17A and 2.8 nM for cynomolgous IL-17A) in the method outlinedin EXAMPLE 5. The IL-17A neutralizing entities described herein werealso tested for species cross-reactivity of neutralizing ability andthough there was a range of neutralizing capabilities, the antagoniststhat worked best to neutralize human IL-17A were also able toeffectively neutralize activity induced by cynomolgous IL-17A. Forexample, in the human SAEC bioassay (described in EXAMPLE 27) cloneM7.19.E7 (SQ7_c87) had an IC50 of 31.1 nM to inhibit human IL-17Ainduced G-CSF production, and this same clone had an IC50 ofapproximately 90 nM to inhibit cynomolgous IL-17A induced G-CSFproduction. Therefore, although the IC50 values were higher againstcynomolgous IL-17A activity compared to human IL-17A, there was clearlyspecies cross-reactivity in the ability of the anti-IL-17A neutralizingentities to inhibit IL-17A-mediated biological activity.

Example 36 Determination of Ability of Anti-Human IL-23 NeutralizingEntities to Cross-React and Neutralize Cynomolgous Monkey IL-23-InducedActivity in IL-23 Bioassays

Similar experiments as described above in EXAMPLE XX (Cynomolgous IL-17Aexperiments) were performed with human versus cynomolgous IL-23, usingIL-23 activity assays described in EXAMPLES 24 and 26, using a fullrange (0-4050 μM) of either recombinant human IL-23 (ZGI lot A1806F) orrecombinant cynomolgous IL-23 (ZGI lot A1922F). Results indicated thatthe activities induced by these two species of IL-23 were identical forthe luciferase-based assay (EC50, in one experiment for example, of 85pM for both species of IL-23) and nearly identical for the pSTAT3-basedassay (46 and 39 pM for human and cynomolgous IL-23 EC50 values,respectively).

In experiments designed to evaluate the ability of the IL-23neutralizing entities described herein to neutralize both human andcynomolgous IL-23, results were similar to those described above forcynomolgous IL-17A: i.e. although there was a range of neutralizingcapacities, those that neutralized human IL-23 best also showed the bestneutralization against cynomolgous IL-23-induced activity. For example,in the IL23R/IL12Rb1 transfectant bioassay (EXAMPLE 24), clone M7.36_B06(SQ22_c305) as a scFv was able to neutralize human IL-23-mediated pSTAT3activity with an IC50 of 0.13 nM and neutralized cynomolgousIL-23-mediated pSTAT3 activity with an IC50 of 0.077 nM. These resultsdemonstrated cynomolgous monkey cross-reactivity for the ability toinhibit IL-23-mediated biological activity.

Example 37 IL-23 Expression Construct

An expression plasmid was constructed via homologous recombination usingtwo DNA fragments, one containing the sequence for pIL12B and onecontaining the sequence for pIL23A, and the expression vector pZMP42.The pIL12B fragment was generated by PCR amplification using primerszc56242 and zc56243. The pIL23A fragment was generated by PCRamplification using primers zc56244 and zc56245.

The pIL12B fragment was made using a previously generated clone ofpIL12B as the template. The fragment includes a 5′ overlap with thepZMP42 vector sequence, the pIL12B segment, and a linker sequencedesigned to join pIL12B to pIL23A. The pIL23A fragment was made using apreviously generated clone of pIL23A as the template. The fragmentincludes a linker sequence designed to join pIL23A to pIL12B, the pIL23Asegment, and a 3′ overlap with the pZMP42 vector sequence. PCRconditions used were as follows: 1 cycle, 94° C., 5 minutes; 35 cycles,94° C., 1 minute, followed by 58° C., 2 minutes, followed by 72° C., 3minutes; 1 cycle, 72° C., 10 minutes.

The PCR reaction mixtures were run on a 1% agarose gel and a bandcorresponding to the sizes of the inserts were gel-extracted using aQIAquick™ Gel Extraction Kit (Qiagen, Cat. No. 28704).

Plasmid pZMP42 is a mammalian expression vector containing an expressioncassette having the MPSV promoter, multiple restriction sites forinsertion of coding sequences, and an otPA signal peptide sequence; aninternal ribosome entry site (IRES) element from Hepatitis C virus, andthe extracellular domain of CD8 truncated at the C-terminal end of thetransmembrane domain; an internal ribosome entry site (IRES) elementfrom poliovirus, a DHFR gene, and the SV40 terminator; an E. coli originof replication; and URA3 and CEN-ARS sequences required for selectionand replication in S. cerevisiae. It was constructed from pZMP21 (PatentPub. No. US 2003/0232414 A1) (deposited at the American Type CultureCollection, 10801 University Boulevard, Manassas, Va. 20110-2209,designated as ATCC #PTA-5266).

The plasmid pZMP42 was cut with BglII prior to recombination in yeastwith the PCR fragment. One hundred microliters of competent yeast (S.cerevisiae) cells were independently combined with 10 μl of the insertDNA and 100 ng of cut pZMP42 vector, and the mix was transferred to a0.2-cm electroporation cuvette. The yeast/DNA mixture was electropulsedusing power supply (BioRad Laboratories, Hercules, Calif.) settings of0.75 kV (5 kV/cm), ∞ ohms, and 25 μF. Six hundred μl of 1.2 M sorbitolwas added to the cuvette, and the yeast was plated in a 100-μl and 300μl aliquot onto two URA-D plates and incubated at 30° C. After about 72hours, the Ura⁺ yeast transformants from a single plate were resuspendedin 1 ml H₂O and spun briefly to pellet the yeast cells. The cell pelletwas resuspended by vortex in 0.1 ml of lysis buffer (2% Triton X-100, 1%SDS, 100 mM NaCl, 10 mM Tris, pH 8.0, 1 mM EDTA), and 0.1 mL of P1 (fromQIAprep Spin Miniprep Kit, Qiagen, cat #27106) with 10 units ofZymolyase added (Zymo Research, cat #E1002). The yeast suspension wasincubated for 10 minutes in a 37° C. waterbath. DNA from the yeast wasisolated using the standard QIAprep Spin Miniprep Kit protocol (Qiagen,cat #27106), starting at the step of adding reagent P2.

Transformation of electrocompetent E. coli host cells (DH12S) was doneusing 5 μl of the yeast DNA prep and 50 μl of cells. The cells wereelectropulsed at 2.0 kV, 25 μF, and 400 ohms. Following electroporation,1 ml SOC (2% Bacto™ Tryptone (Difco, Detroit, Mich.), 0.5% yeast extract(Difco), 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl₂, 10 mM MgSO₄, 20 mMglucose) was added and then the cells were plated in a 50 μl and a 200μl aliquot on two LB AMP plates (LB broth (Lennox), 1.8% Bacto™ Agar(Difco), 100 mg/L Ampicillin).

The inserts of five clones for the construct were subjected to sequenceanalysis and one clone, containing the correct sequence, was selected.Larger scale plasmid DNA was isolated using a commercially available kit(QIAGEN Plasmid Mega Kit, Qiagen, Valencia, Calif.) according tomanufacturer's instructions.

Three sets of 200 μg of the zCyto38f2 construct were each digested with200 units of Pvu I at 37° C. for three hours and then were precipitatedwith IPA and spun down in a 1.5 mL microfuge tube. The supernatant wasdecanted off the pellet, and the pellet was washed with 1 mL of 70%ethanol and allowed to incubate for 5 minutes at room temperature. Thetube was spun in a microfuge for 10 minutes at 14,000 RPM and thesupernatant was decanted off the pellet. The pellet was then resuspendedin 750 μl of ZF1 media in a sterile environment, allowed to incubate at60° C. for 10 minutes, and was allowed to cool to room temperature. 5E65×SA cells were spun down in each of three tubes and were resuspendedusing the DNA-media solution. The DNA/cell mixtures were placed in a 0.4cm gap cuvette and electroporated using the following parameters: 950μF, high capacitance, and 300 V. The contents of the cuvettes were thenremoved, pooled, and diluted to 25 mLs with ZF1 media and placed in a125 mL shake flask. The flask was placed in an incubator on a shaker at37° C., 6% CO₂, and shaking at 120 RPM.

The cell line was subjected to nutrient selection followed by stepamplification to 200 nM methotrexate (MTX). Expression was confirmed bywestern blot, and the cell line was scaled-up and protein purificationfollowed.

Example 38 Expression of IL23 C-Terminal His Tagged Protein in CHO DXB11Cells in a Wave Reactor

IL23 v.1 CH6 protein (IL23A/IL12B) was expressed in a 20 L WavebagReactor (Wave Biotech) in CHO DXB11 cells transfected with the ZGconstruct 1564. The cells were scaled up in shake flasks using ZF1medium (JRH imMEDiAte Advantage Cat #65633) with the addition of 5 mML-glutamine (from 200 mM L-glutamine, Gibco catalog 425030-081), 1 mMsodium pyruvate (from 100 mM Sodium Pyruvate, Gibco catalog #11360-070)and 500 nM methotrexate. The reactor run was initiated by seeding 1.7 Lof shake flask culture in log phase into 8.3 L ZF1 medium containingL-glutamine and sodium pyruvate but no methotrexate. This resulted in a10 L final working volume with a density of 3.1×10E5 c/mL.

The CO₂ level was maintained at 6% and was pumped continually into theheadspace of the reactor at 0.1 LPM. Dissolved oxygen requirements ofthe cells were met by rocking the culture on a platform at a rate of 25rocks per minute at an angle setting of 9.5. pH was not controlled butstayed between 6.6 and 6.9. Temperature was maintained at 37° C. untildensity reached 7.5×10E5 cells/mL, then temperature was dropped to 34°C. for the remainder of the run. Glucose levels were maintained above 2g/L and L-glutamine above 2 mM.

The culture was harvested 9 days after seeding with a density of4.7×10E6 cells/mL and 97% viability. The supernatant was centrifuged at3500×g for 15 minutes and the clarified conditioned medium was passedthrough a 0.22 μm filter (Millipore Opticap Cat #KW1904HB3) andsubmitted for protein purification.

Example 39 Transfection and Expression of Cyno IL23 Protein in 293 Cells

Cynomolgus IL23 was produced transiently in 293F cells (Invitrogen,Carlsbad, Calif. Cat #R790-07). Large-scale plasmid DNA was isolatedusing a PureLink HiPure Plasmid Gigaprep Kit (Invitrogen, Carlsbad,Calif. Cat #K210009) according to manufacturer's instructions. 293Fsuspension cells were cultured in 293 Freestyle medium (Invitrogen,Carlsbad, Calif. Cat #12338-018) at 37° C., 6% CO2 in four 3 L spinnerflasks at 95 RPM. Fresh medium was added to each spinner immediatelyprior to transfection to obtain a 4.5 liter working volume at a finaldensity of 1×10E6 cells/mL. For each spinner, 2.0 mL of Lipofectamine2000 (Invitrogen, Carlsbad, Calif. Cat #11668-019) was added to 20 mLOpti-MEM medium (Invitrogen, Carlsbad, Calif. Cat #31985-070) and 1.5 mgof DNA (ZG Construct 1630) was diluted in a separate tube of 20 mLOpti-MEM. For each spinner, the DNA and lipid were incubated separatelyat room temperature for 5 minutes, then combined and incubated togetherfor an additional 30 minutes at room temperature with occasional gentlemixing. One tube of lipid-DNA mixture was added to each spinner of 293Fcells which was returned to 37° C., 6% CO2 at 75 RPM. Afterapproximately 96 hours, the conditioned medium was harvested, 0.2 μMfiltered and submitted for protein purification.

Example 40 IL-23 Protein Purification A) Purification of Human IL-23Preparation of Human IL23 v.1 for IMAC Capture—

˜10 L of 1× media were augmented to 0.02% sodium azide from 1000× stockand put through a UF/DF process wherein the media was first concentrated10× against 3×0.1 m² 10 kD Pellicon membranes (Millipore) in aperistaltic pump system. Then the concentrate was diafiltered intophosphate buffered saline via 5 system volume exchanges. Harvested UF/DFmedia was adjusted to 0.5M NaCl (via addition of 0.4M Solid), 25 mMImidazole (addition of solid) and the pH adjusted to 7.5 (using HCl).The adjusted concentrate was 0.22 um sterile filtered (Nalgene) andanalyzed via RP-HPLC and Western blot for recovery, comparing against 1×media and the permeates. Analyses show that target is nearly completelyrecovered at this step.

IMAC (Immobilized Metal Affinity Chromatography) Capture—

UF/DF media was loaded over 137 mL Ni NTA His Bind Resin (Novagen)packed in a 2 cm glass column (Millipore) at 0.2 mL/min at 4 C. Thecolumn was equilibrated in 500 mM NaCl, 50 mM NaPO₄, 25 mM Imidazole pH7.5 buffer. Upon complete washout of the applied media, the flow ratewas increased to 20 mL/min and the column washed with equilibrationbuffer until UV at 254 nm and 280 nm was baseline stable. Bound proteinwas eluted stepwise using a 40 mM and 500 mM Imidazole steps, each inequilibration buffer. The elution flow rate was 20 mL/min and 25 mLfractions were collected. Pools were made based on the inflection ofA280 nm and analyzed by RP-HPLC, as well as reducing and non-reducingSDS-PAGE coomassie gels. Only the 500 mM pool had target as analyzed bythese methods. Protein was completely captured by the IMAC resin, asassessed by Western blot and RP-HPLC.

Superdex 200 Coarse SEC (Size Exclusion Chromatography)

The 500 mM IMAC pool was considered pure enough to warrant SEC for finalformulation and polishing. The pool was initially concentrated to 50 mLagainst 1×50 cm²-100 kD MWCO membrane (Millipore) in the Labscale TFFsystem. At 50 mL, this concentrate was transferred to a 30 kD Ultracelmembrane (Millipore). Concentration continued via centrifugation until afinal volume of 15 mL was reached. The final concentrate was injectedover a 26/60 (318 mL) Superdex 200 size exclusion column (GE Healthcare)running in 35 mM NaPO₄, 120 mM NaCl pH 7.2 at 3.0 mL/min. 2.5 mLfractions were collected throughout the isocratic elution. A total ofthree runs were performed, with 6 mL injections per run. Elutionfractions were analyzed by SDS-PAGE non-reducing gel, and the resultingpools were analyzed by RP-HPLC. Monomeric protein was selectivelypooled.

Superdex 200 Fine SEC (Size Exclusion Chromatography)

The pools of protein from the first size exclusion run were slightlycontaminated with higher molecular weight species. They were pooledtogether, and re-concentrated to <10 mL against 63.5 mm YM30 stirredcell membrane (Millipore). The concentrate was re-injected over theSuperdex 200 size exclusion column under identical conditions asprevious. A conservative pool was made based on the UV absorbanceinflection at 280 nm, and assayed by RP-HPLC for a putative targetconcentration. This final pool was 0.22 um filtered (Millipore),aliquoted, and stored at −80 C.

B) Purification of Cyno IL23 v.4 from 293F Transient Host—IMAC Capture—

˜5.8 L of delivered media was concentrated to <1 L against 1×0.1 m2 MWCOpellicon membrane (Millipore) using a peristaltic pump system. Theconductivity of the concentrated media was adjusted to be equivalent tothat of 0.5M NaCl via addition of 0.4M solid reagent, 25 mM Imidazolevia addition of solid, pH 7.5 using HCl and loaded over 5 mL NiSepharose 6 FF resin (GE Healthcare) packed in a 1 cm diameter glasscolumn (Millipore) at 0.9 mL/min overnight at 4 C. Upon depletion ofmedia, flow rate increased to 4 mL/min and the column washed with 50 mMNaPO4, 500 mM NaCl, 25 mM Imidazole pH 7.5 until UV at A254 nm and A280nm baseline stable. Bound target eluted using steps of 40 mM and 500 mMImidazole in the above mentioned equilibration buffer. Elution proceededat 2 mL/min, collecting 3 mL fractions throughout. Pooling of proteinwas based on the A280 nm inflection of the 500 mM step, and pool targetconcentration was analyzed via RP-HPLC.

Superdex 200 SEC

500 mM Imidazole Pool considered pure enough to warrant size exclusionchromatography for final formulation and polishing. The Ni Sepharoseprotein pool was concentrated to <3.0 mL against a 30 kD MWCO Ultracelmembrane (Millipore). The concentrate was injected over 16/60 (120 mL)Superdex 200 size exclusion column (GE Healthcare) equilibrated in 35 mMNaPO4, 120 mM NaCl pH 7.2 at 1.02 mL/min flowrate. Fractions (1.0 mL)were collected throughout the isocratic elution. Conservative poolingwas based on the inflection of the A280 nm signal. The size exclusionpool was concentrated to 2 mL using another 30 kD MWCO Ultracelmembrane, 0.22 um filtered (Millipore), aliquotted, and stored at −80 C.

Example 41 Expression of IL23R Fc-Tagged Protein in CHO DXB11 Cells in aWave Reactor

IL23R Fc5 protein was expressed in a 20 L Wavebag Reactor (Wave Biotech)in CHO DXB11 cells transfected with the ZG construct 1602. The cellswere scaled up in shake flasks using ZF1 medium (JRH imMEDIAte AdvantageCat #65633) with the addition of 5 mM L-glutamine (from 200 mML-glutamine, Gibco catalog #25030-081), 1 mM sodium pyruvate (from 100mM Sodium Pyruvate, Gibco catalog #11360-070) and 500 nM methotrexate.The reactor run was initiated by seeding 900 mL of shake flask culturein log phase into 9.1 L ZF1 medium containing L-glutamine and sodiumpyruvate but no methotrexate. This resulted in a 10 L final workingvolume with a density of 2.0×10E5 c/mL.

The CO₂ level was maintained at 6% and was pumped continually into theheadspace of the reactor at 0.1 LPM. Dissolved oxygen requirements ofthe cells were met by rocking the culture on a platform at a rate of 25rocks per minute at an angle setting of 9.5. pH was not controlled.Temperature was maintained at 37° C. until density reached approximately8×10E5 cells/mL, then temperature was dropped to 34° C. for theremainder of the run. Glucose levels were maintained above 2 g/L andL-glutamine above 2 mM.

The culture was harvested 9 days after seeding with a density of8.0×10E6 cells/mL and 98% viability. The supernatant was centrifuged at3500×g for 15 minutes and the clarified conditioned medium was passedthrough a 0.22 um filter (Millipore Opticap Cat #KW1904HB3) andsubmitted for protein purification.

Example 42 Purification of Human IL23R-Fc5 from CHO Cells

Recombinant carboxyl terminal Fc5 tagged human IL23R was produced fromtransfected CHO cells expressing target at 12 mg/L. The CHOtransfections were performed using methods known in the art. 10 L ofconditioned media were harvested, sterile filtered using 0.2 μm filtersand adjusted to pH 7.4. The protein was purified from the filtered mediausing a combination of POROS® A50 Protein A Affinity Chromatography(Applied Biosciences, Foster City, Calif.) and Superdex 200 SizeExclusion Chromatography (GE Healthcare, Piscataway, N.J.) A 27 mlPOROS® A50 column (20 mm×85 mm) was pre-eluted with 3 column volumes(CV) of 25 mM Citrate-Phosphate (1.61 mM Sodium Citrate-23.4 mM SodiumPhosphate,) 250 mM Ammonium Sulfate pH 3 buffer and equilibrated with 20CV PBS. Direct loading to the column at 8 ml/min overnight at 4° C.captured the IL23R-Fc5 in the conditioned media. After loading wascomplete, the column was washed with 10 CV of equilibration buffer. Nextthe column was washed with 10 CV of 25 mM Citrate-Phosphate, 250 mMAmmonium Sulfate pH 7.4 buffer following which the bound protein waseluted at 10 ml/min with a 5 CV gradient from pH 7.4 to pH 3 formedusing the Citrate Phosphate-Ammonium Sulfate buffers. Fractions of 5.0ml each were collected into tubes containing 500 μl of 2.0 M Tris, pH8.0 and mixed immediately in order to neutralize the eluted proteins.The fractions were pooled based on A280 and non-reducing SDS-PAGE.

The IL23R-Fc5-containing pool was concentrated to 10 ml byultrafiltration using Amicon Ultra-15 30K NWML centrifugal devices(Millipore), and injected onto a 318 ml (26 mm×600 mm) Superdex 200column pre-equilibrated in 35 mM Sodium Phosphate, 120 mM NaCl pH 7.3 at28 cm/hr. The fractions containing purified. IL23R-Fc5 were pooled basedon A280 and SDS PAGE, filtered through a 0.2 μm filter and frozen asaliquots at −80° C. The concentration of the final purified protein wasdetermined by BCA assay (Pierce, Rockford, Ill.). The overall processrecovery was approximately 66%.

Analysis of Purified IL23R-Fc5

Recombinant IL23R-Fc5 was analyzed by SDS-PAGE (4-12% BisTris,Invitrogen, Carlsbad, Calif.) with 0.1% Coomassie 8250 staining forprotein and immunoblotting with Anti-IgG-HRP. The purified protein waselectrophoresed and transferred to nitrocellulose (0.2 μm; Invitrogen,Carlsbad, Calif.) at ambient temperature at 600 mA for 45 minutes in abuffer containing 25 mM Tris base, 200 mM glycine, and 20% methanol. Thefilters were then blocked with 10% non-fat dry milk in 50 mM Tris, 150mM NaCl, 5 mM EDTA, 0.05% Nepal (TBS) for 15 minutes at roomtemperature. The nitrocellulose was quickly rinsed, and the IgG-HRPantibody (1:10,000) was added. The blots were incubated overnight at 4°C., with gentle shaking. Following the incubation, the blots were washedthree times for 10 minutes each in TBS, and then quickly rinsed in H₂O.The blots were developed using commercially available chemiluminescentsubstrate reagents (Roche LumiLight), and the signal was captured usingLumi-Imager's Lumi Analyst 3.0 software (Boehringer Mannheim GmbH,Germany.) The purified IL23R-Fc5 appeared as a band at about 200 kDA onboth the non-reducing Coomassie stained gel and on the immunoblot,suggesting a glycosylated dimeric form as expected. The protein had thecorrect NH₂ terminus and the correct amino acid composition.

Example 43 Cynomolgus IL17A CH6 Expression in 293F

An expression plasmid encoding cynomolgus IL17A CH6 was constructed viahomologous recombination in yeast with a DNA fragment containing theIL17A CH6 sequence.

A cDNA fragment was created using PCR and includes residues 1-465. Theupstream primer consists of 41 bases of overlap with the vector backboneand 24 bases of the 5′ end open reading from of the amino terminus ofthe inserted gene. The downstream primer contains 37 residues of overlapwith the backbone vector, the His tag with a GSGG linker, and 23 basesof the gene.

Amino acid 127 was mutated from R to P to eliminate a potential cleavagesite. This mutation converts the cynomolgus sequence to the humansequence at this position. The mutation was created by overlapping twofragments via PCR. zc57312, as explained above, was used with a 24 basedownstream primer with its 5′ end at amino acid 127 and its 3′ end 134.This created the 5′ fragment of the gene containing the mutation. The 3′fragment was constructed using a forward primer containing the entiresequence from amino acids 126 to 155. This was overlapped with primerzc57313, described previously. These two fragments were fused via PCR.

The PCR amplification conditions, using Promega's GoTaq (Promega,Madison, Wis., catalog #M712B), were as follows: 1 cycle of 94° C., 5minutes; 29 cycles of 94° C. for 1 minute, 55° C. for 30 seconds, 68° C.for 30 seconds; 1 cycle of 4° C. forever. The entire product of each PCRwas run on a 1% LMP agarose gel (Seaplaque GTG) with 1×TAE buffer foranalysis. After excising the appropriate band, it was purified usingQiagen's gel purification kit (Qiagen, Valencia, Calif., catalog#28704).

One hundred μL of competent yeast cells (S. cerevisiae) was combinedwith 10 μl of purified DNA from above, mixed with 100 ng of BglII-cutplasmid, and transferred to a 0.2 cm electroporation cuvette. Theyeast-DNA mixture was electropulsed at 0.75 kV (5 kV/cm), ∞ ohms, 25 μF.To each cuvette was added 600 μl of 1.2M sorbitol, and the yeast wereplated onto a URA-DS plate and incubated at 30° C. After about 72 hours,approximately 504 packed yeast cells taken from the Ura+ yeasttransformants of a single plate was resuspended in 100 μL of lysisbuffer (2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris, pH 8.0, 1 mMEDTA), 100 μL of Qiagen P1 buffer from a Qiagen miniprep kit (Qiagen,Valencia, Calif., catalog #27104), and 20 U of Zymolyase (Zymo Research,Orange, Calif., catalog #1001). This mixture was incubated for 30minutes at 37° C., and the remainder of the Qiagen miniprep protocol wasperformed, using 30 uL buffer EB for elution.

Forty μL electrocompetent E. coli cells (DH12S, Invitrogen, Carlsbad,Calif.) was transformed with 3 μL yeast DNA in a 0.2 cm electroporationcuvette. The cells were electropulsed at 1.75 kV, 25 μF., and 400 ohms.Following electroporation, 600 μL SOC (2% Bacto' Tryptone (Difco,Detroit, Mich.), 0.5% yeast extract (Difco), 10 mM NaCl, 2.5 mM KCl, 10mM MgCl2, 10 mM MgSO4, 20 mM glucose) was added to the cuvette. 10 μL ofthis solution was plated on an LB AMP plate (LB broth (Lennox), 1.8%Bacto Agar (Difco), 100 mg/L Ampicillin).

Individual clones harboring the correct expression construct forcynomolgus IL17A CH6 were identified by restriction digest with 20 UFspI and 20 U PvuII to verify the presence of the insert. The inserts ofpositive clones were subjected to sequence analysis. Larger scaleplasmid DNA was isolated using the Invitrogen mega prep kit (Invitrogen,Carlsbad, Calif., catalog #457009) according to manufacturer'sinstructions.

Transfection into 293F Cells:

To test for expression of the cynomolgus IL17A CH6 protein, 293F cellswere transiently transfected using Lipofectamine-2000 (Invitrogen,Carlsbad, Calif., catalog #11668-019) and OptiMEM (Invitrogen, Carlsbad,Calif., catalog #31985-070) and grown in a 12-well plate. 1 μg plasmidDNA and one million cells were used for the transfection. After 96hours, medium was harvested and prepared for a Western blot assay.

Invitrogen materials and protocols were used for the Western blot withanti-6× histidine (R&D Systems, Minneapolis, Minn., catalog #MAB050H) asthe detection antibody. Significant expression was observed, so a largescale transfection was done for protein acquisition.

Six 1000 mL flasks were seeded with 250 mL 293F cells at one millioncells per mL. 20 mL OptiMEM was placed in each of two 50 mL conicaltubes. 2 mL Lipofectamine-2000 was added to one 50 mL conical tube and1.5 mg of the cynomolgus IL17A CH6 expression plasmid was placed in theother tube. The tubes were inverted several times and allowed toincubate for 5 minutes at room temperature. The two tubes were thenmixed together, inverted several times, and allowed to incubate for 30minutes at room temperature. While swirling the cells, theDNA-Lipofectamine-2000 mixture was evenly distributed into each of thesix flasks. The flasks were then incubated on a shaker at 37° C., 6%CO₂, and shaken at 120 rpm. Cells were harvested 96 hours later.

The DNA sequence is shown in SEQ ID NO:1018. The polypeptide sequence isshown in SEQ ID NO: 1019.

Example 44 Transfection and Expression of Cyno IL17A C-TerminalHis-Tagged Protein in 293 Cells

Cynomolgus IL17A R127P CH6 was produced transiently in 293F cells(Invitrogen, Carlsbad, Calif. Cat #R790-07). Briefly, 293F suspensioncells were cultured in 293 Freestyle medium (Invitrogen, Carlsbad,Calif. Cat #12338-018) at 37° C., 6% CO2 in a 3 L spinner flask at 95RPM. Fresh medium was added immediately prior to transfection to obtaina one liter working volume at a final density of 1×10E6 cells/ml. Foreach spinner, 1.3 mL of Lipofectamine 2000 (Invitrogen, Carlsbad, Calif.Cat #11668-019) was added to 15 mL Opti-MEM medium (Invitrogen,Carlsbad, Calif. Cat #31985-070) and 1.0 mg of DNA (ZG Construct 1623)was diluted in a separate tube of 15 mL Opti-MEM. Each tube wasincubated separately at room temperature for 5 minutes, then combinedand incubated together for an additional 30 minutes at room temperaturewith occasional gentle mixing. The lipid-DNA mixture was then added tothe spinner of 293F cells which was returned to 37° C., 6% CO₂ at 75RPM. After approximately 96 hours, the conditioned medium was harvested,0.2 μM filtered and submitted for protein purification.

Example 45 Cynomolgus IL17F CH6 Expression in 293F

An expression plasmid encoding cynomolgus IL17F CH6 was constructed viahomologous recombination in yeast with a DNA fragment containing theIL17F CH6 sequence.

A cDNA fragment was created using PCR and includes residues 1-522. Theupstream primer consists of 42 bases of overlap with the vector backboneand 24 bases of the 5′ end open reading from of the amino terminus ofthe inserted gene. The downstream primer contains 37 residues of overlapwith the backbone vector, the His tag with a GSGG linker, and 23 basesof the gene.

The PCR amplification conditions, using Promega's GoTaq (Promega,Madison, Wis., catalog #M712B), were as follows: 1 cycle of 94° C., 5minutes; 29 cycles of 94° C. for 1 minute, 55° C. for 30 seconds, 68° C.for 30 seconds; 1 cycle of 4° C. forever. The entire product of each PCRwas run on a 1% LMP agarose gel (Seaplaque GTG) with 1×TAE buffer foranalysis. After excising the appropriate band, it was purified usingQiagen's gel purification kit (Qiagen, Valencia, Calif., catalog#28704).

One hundred μL of competent yeast cells (S. cerevisiae) was combinedwith 10 μl of purified DNA from above, mixed with 100 ng of BglII-cutplasmid, and transferred to a 0.2 cm electroporation cuvette. Theyeast-DNA mixture was electropulsed at 0.75 kV (5 kV/cm), ∞ ohms, 25 μF.To each cuvette was added 600 μl of 1.2M sorbitol, and the yeast wereplated onto a URA-DS plate and incubated at 30° C. After about 72 hours,approximately 50 μL packed yeast cells taken from the Ura+ yeasttransformants of a single plate was resuspended in 100 μL of lysisbuffer (2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris, pH 8.0, 1 mMEDTA), 100 μL of Qiagen P1 buffer from a Qiagen miniprep kit (Qiagen,Valencia, Calif., catalog #27104), and 20 U of Zymolyase (Zymo Research,Orange, Calif., catalog #1001). This mixture was incubated for 30minutes at 37° C., and the remainder of the Qiagen miniprep protocol wasperformed, using 30 uL buffer EB for elution.

Forty μL electrocompetent E. coli cells (DH12S, Invitrogen, Carlsbad,Calif.) was transformed with 3 μL yeast DNA in a 0.2 cm electroporationcuvette. The cells were electropulsed at 1.75 kV, 25 μF, and 400 ohms.Following electroporation, 600 μL SOC (2% Bacto' Tryptone (Difco,Detroit, Mich.), 0.5% yeast extract (Difco), 10 mM NaCl, 2.5 mM KCl, 10mM MgCl2, 10 mM MgSO4, 20 mM glucose) was added to the cuvette. 10 μL ofthis solution was plated on an LB AMP plate (LB broth (Lennox), 1.8%Bacto Agar (Difco), 100 mg/L Ampicillin).

Individual clones harboring the correct expression construct forcynomolgus IL17F CH6 were identified by restriction digest with 20 UFspI and 20 U BsrGI to verify the presence of the insert. The inserts ofpositive clones were subjected to sequence analysis. Larger scaleplasmid DNA was isolated using the Invitrogen mega prep kit (Invitrogen,Carlsbad, Calif., catalog #457009) according to manufacturer'sinstructions.

Transfection into 293F Cells:

To test for expression of the cynomolgus IL17F CH6 protein, 293F cellswere transiently transfected using Lipofectamine-2000 (Invitrogen,Carlsbad, Calif., catalog #11668-019) and OptiMEM (Invitrogen, Carlsbad,Calif., catalog #31985-070) and grown in a 12-well plate. 1 ug plasmidDNA and one million cells were used for the transfection. After 96hours, medium was harvested and prepared for a Western blot assay.

Invitrogen materials and protocols were used for the Western blot withanti-6× histidine (R&D Systems, Minneapolis, Minn., catalog #MAB050H) asthe detection antibody. Significant expression was observed, so a largescale transfection was done for protein acquisition.

Six 1000 mL flasks were seeded with 250 mL 293F cells at one millioncells per mL. 20 mL OptiMEM was placed in each of two 50 mL conicaltubes. 2 mL Lipofectamine-2000 was added to one 50 mL conical tube and1.5 mg of the cynomolgus IL17F CH6 expression plasmid was placed in theother tube. The tubes were inverted several times and allowed toincubate for 5 minutes at room temperature. The two tubes were thenmixed together, inverted several times, and allowed to incubate for 30minutes at room temperature. While swirling the cells, theDNA-Lipofectamine-2000 mixture was evenly distributed into each of thesix flasks. The flasks were then incubated on a shaker at 37° C., 6%CO₂, and shaken at 120 rpm. Cells were harvested 96 hours later.

The DNA sequence is shown in SEQ ID NO:1020. The polypeptide sequence isshown SEQ ID NO: 1021.

Example 46 Transfection and Expression of Cyno IL17F C-TerminalHis-Tagged Protein in 293 Cells

Cynomolgus IL17F CH6 was produced transiently in 293F cells (Invitrogen,Carlsbad, Calif. Cat #R790-07). Briefly, 293F suspension cells werecultured in 293 Freestyle medium (Invitrogen, Carlsbad, Calif. Cat#12338-018) at 37° C., 6% CO2 in a 3 L spinner flask at 95 RPM. Freshmedium was added immediately prior to transfection to obtain a one literworking volume at a final density of 1×10E6 cells/mL. For each spinner,1.3 mL of Lipofectamine 2000 (Invitrogen, Carlsbad, Calif. Cat#1.1668-019) was added to 15 mL Opti-MEM medium (Invitrogen, Carlsbad,Calif. Cat #31985-070) and 1.0 mg of DNA (ZG Construct 1628) was dilutedin a separate tube of 15 mL Opti-MEM. Each tube was incubated separatelyat room temperature for 5 minutes, then combined and incubated togetherfor an additional 30 minutes at room temperature with occasional gentlemixing. The lipid-DNA mixture was then added to the spinner of 293Fcells which was returned to 37° C., 6% CO2 at 75 RPM. Afterapproximately 96 hours, the conditioned medium was harvested, 0.2 μMfiltered and submitted for protein purification.

Example 47 Purification of cyno IL17A R127P CH6 and Cyno IL17F CH6 NiIMAC Capture—

Delivered media was adjusted to 0.5M NaCl and 25 mM Imidazole viaaddition of solid, pH 7.5 via slowly adding 10N NaOH while stirring.Adjusted media was loaded over 5 mL Ni Sepharose 6 FF resin (GEHealthcare) packed in a 1.0 cm diameter column (Millipore) at 0.9 mL/minovernight at 4 C. Upon depletion of media, the flow rate was increasedto 4 mL/min and the column washed with 50 mM NaPO4, 500 mM NaCl, 25 mMImidazole pH 7.5 until UV@A254 nm and A280 nm was baseline stable. Boundtarget was eluted using steps of 40 mM and 500 mM Imidazole in the abovementioned equilibration buffer. Elution proceeded at 2 mL/min,collecting 3 mL fractions throughout. Only fractions from the 500 mMImidazole step contained target, a pool was made based on the 280 nmabsorbance inflection and analyzed by RP HPLC. By analytical RP-HPLC,the entire expressed CH6 target was captured on the IMAC resin.

Superdex 75 Size Exclusion Chromatography:

500 mM Imidazole Pool was considered pure enough to warrant sizeexclusion chromatography for final formulation. The 500 mM ImidazoleIMAC pool was concentrated to <5.0 mL using a 10 kD MWCO IA Ultracelmembrane (Millipore). The concentrate was injected over a 26/60 (318 mL)Superdex 75 SEC column (GE Healthcare) equilibrated in 35 mM NaPO4, 120mM NaCl pH 7.2. at a flow rate of 3.0 mL/min while collecting 2.5 mLfractions throughout the isocratic elution. Conservative pooling wasbased on the inflection of A280 nm signal and SDS-PAGE analysis ofcollected fractions. The size exclusion pool was concentrated to 1 mg/mLusing another 10 kD MWCO Ultracel membrane, 0.22 um filtered(Millipore), aliquotted, and stored at −80 C.

Example 48 Expression of Human IL17RA Fc-Tagged Protein in CHO DXB11Cells in a Wave Reactor

IL17RA Fc5 (aka IL17R[FL]x1 C Fc5) protein was expressed in a 20 LWavebag Reactor (Wave Biotech) in CHO DXB11 cells transfected with theZG construct 1466. The cells were scaled up in shake flasks using ZF1medium (JRH imMEDiAte Advantage Cat #65633) with the addition of 5 mML-glutamine (from 200 mM L-glutamine, Gibco catalog #25030-081), 1 mMsodium pyruvate (from 100 mM Sodium Pyruvate, Gibco catalog #11360-070)and 500 nM methotrexate. The reactor run was initiated by seeding oneliter of shake flask culture in log phase into 9 L ZF1 medium containingL-glutamine and sodium pyruvate but no methotrexate. This resulted in a10 L final working volume with a density of 1.7×10E5 c/mL.

The CO₂ level was maintained at 6% and was pumped continually into theheadspace of the reactor at 0.1 LPM. Dissolved oxygen requirements ofthe cells were met by rocking the culture on a platform at a rate of 25rocks per minute at an angle setting of 9.5. pH was not controlled butstayed between 6.6 and 7.0. Temperature was maintained at 37° C. untildensity reached approximately 1×10E6 cells/mL, then temperature wasdropped to 34° C. for the remainder of the run. Glucose levels weremaintained above 2 g/L and L-glutamine above 2 mM.

The culture was harvested 12 days after seeding with a density of6.3×10E6 cells/mL and 98% viability. The supernatant was centrifuged at3500×g for 15 minutes and the clarified conditioned medium was passedthrough a 0.22 μm filter (Millipore Opticap Cat # KW1904HB3) andsubmitted for protein purification.

Example 49 Purification of Human IL17R [FL][x1] C(Fc5) from CHO DXB115×SA Media Poros Protein A 50 Affinity Column Capture—

Expression of Fc5 tagged target in delivered media was assessed via ananalytical protein A-HPLC assay. Upon determining the expression levels,an appropriate volume of Poros A50 resin used to capture the expressedtarget, assuming a binding capacity of ˜9 mg target per mL of packedbed. 10 L of delivered media were captured on 2×2 cm glass columns(Millipore), each column being 50 mL in bed volume. Each of the columnswas packed with 50 mL Poros A50 resin (AB Biosystems) and had beenequilibrated in the wash buffer indicated below. After the entire volumeof media loaded over the resin, the columns were washed with 1.6 mMCitrate-monohydrate, 10.9 mM dibasic sodium phosphate, 0.25M AmmoniumSulfate, pH 6.0 until UV at A280 nm baseline stable. Bound protein waseluted from the resin via a 60 CV gradient from the above wash bufferto; 20 mM citrate-monohydrate, 5 mM dibasic sodium phosphate, 0.25MAmmonium Sulfate, pH 3.0 at 20 mL/min. Fractions were collected andneutralized using enough 2M. Tris pH 8.0 to deliver a final pH of 7.0.The elution pool was analyzed via the above mentioned analytical proteinA-HPLC assay.

Concentration of Protein a Affinity Column Pool—

Initial concentration of protein A affinity pools was performed using1×50 cm²10 kD MWCO Biomax membrane (Millipore) set up in a Labscale TFFsystem (Millipore). Once the volume reached 120 mL, the concentrate wastransferred to a stirred cell system (Millipore) with a 10 kD PESmembrane (Millipore). Concentration was continued to a final volume of20 mL. The stirred cell permeates were analyzed for Fc tagged target viathe above mentioned analytical protein A-HPLC assay.

Superdex 200 Size Exclusion Chromatography—

2×10 mL volumes of the concentrated protein A affinity pool wereinjected over a 340 mL, XK26 glass Superdex 200 SEC column (GEHealthcare) at a flow rate of 3.5 mL/min. The column was equilibrated inthe following mobile phase; 35 mM NaPO4, 120 mM NaCl, pH 7.2. Fractionscontaining Fc tagged target were pooled, 0.22 um filtered (Millipore),aliquoted, and stored at −80 C.

Example 50 Preparation and Extraction of Insoluble IL17A fromEscherichia coli

E. coli W3110 cells from a 2 L bioreactor fermentation containing thehuman IL17A were washed with 50 mM Tris pH 8 containing 200 mM NaCl and5 mM EDTA to remove any broth contaminants. 1.2 L of ice cold lysisbuffer (50 mM Tris pH 8, 200 mM NaCl, 5 mM EDTA, 5 mM Benzamidine and 5mM DTT) was added to the 323 g cell pellet and homogenized using thePolytron tissue-grinder until all clumps were disrupted. Next, thebacterial cells were lysed with three passes through a MicroFluidizerkeeping the cell suspension chilled to 4° C. The final volume of thecell lysate was 1.73 L. The inclusion bodies in this lysate werepelleted by centrifuging 30 min at 20,000×g (12,000 rpm in a JA-14 rotorin a Beckman J2-21M centrifuge), 4° C. in eight 250-ml centrifugebottles. The inclusion bodies were washed three times with lysis bufferto remove any E. coli unbroken cells and large cellular debris from thepelleted inclusion body protein. The supernatant was carefully pouredoff the pellet and the inclusion bodies were washed by suspending thepellet in lysis buffer and completely homogenizing to wash out solubleproteins and cellular components. The washed inclusion bodies wererecovered by centrifuging 30 min at 15,000×g (12,000 rpm in JA-14), 4°C. Three of four washed pellets (38 g each) were stored at −80° C. in250 ml bottles and the fourth bottle was processed further. 7MGuanidine-HCl in 50 mM Tris pH 8 containing 100 mM sodium sulfite and 20mM sodium tethrathionate was used to extract recombinant protein fromwashed pellets. Extraction with the denaturant dissociatesprotein-protein interactions and unfolds the protein. As a result, theextracted protein consists of unfolded monomers, with sulfhydryl groupsin the reduced state and sulphonated. Using the tissue homogenizer, the38 g pellet was homogenized with 150 ml extraction buffer and clarifiedby centrifuging the suspension for two hours at 35,000×g at 4° C. The178 ml clarified extract was evaluated by RP HPLC (23.3 mg/ml) and SDSPAGE (20.4 mg/ml) and divided into four parts. Three parts were storedat −80° C. until required. The fourth part was used for extract forpreparing folded IL17A.

Refolding:

Ice cold refolding buffer (4 Liters) of the following composition wasprepared; 0.75M Arginine, 55 mM MES (N-MorpholinoEthaneSulfonic acid),10.56 mM NaCl, 0.44 mM KCl, 0.055% Peg 3.4 K (w/v), 1.1 mM EDTA, 440 mMSucrose, 550 mM GuHCl, 1 mM GSH (reduced Glutithione), 1 mM GSSG(oxidized Glutithione), pH 6.5

The oxidation-reduction pair (GSH:GSSG) was added just prior to dilutingthe S-sulphonated inclusion body stock. The concentration of theSulfytolized inclusion bodies was 20.4 mg/mL as determined by RP HPLCusing suitable IL17F standard. 20 mL of the concentrated stock wasadded, dropwise, to 4 Liters of well stirred ice cold refolding buffer.Upon completing the dilution process, the atmosphere was flooded withNitrogen and the vessel was tightly capped and placed in the cold roomwith gentle stirring. At various time intervals, samples were withdrawnfor RP HPLC analysis. Each time a sample was taken, the vessel wasflooded with Nitrogen gas and tightly capped, and replaced in the coldroom with gentle stirring.

The observed HPLC dynamics at earliest points show multiple clusteredpeaks with earlier elution times than that of the starting material.Minimal peaks were observed downstream of the staring material elutiontime. Over time, a downfield peak began to increase at the expense ofthe upstream cluster of peaks. The cluster represents numerous partiallyGlutithyiolated moieties. The dimeric product peak advanced slowly overtime at the expense of the up stream peak cluster.

Capture and Recovery on Cation Exchange (CIEX):

A cation exchange column (63 ml bed volume, 2 cm. Diameter) of SP FastFlow (GE Healthcare) was equilibrated in Buffer A: 25 mM Acetic Acid,100 mM NaCl, at pH 5.0. Two other buffers were utilized to run the CIEXprocess: Buffer B: 25 mM Acetic Acid; 2 Molar NaCl, pH 5.0 and DilutionBuffer: 100 mM Acetic acid, pH 4.7

The capture strategy utilizes in-line dilution proportioning at 20%refold Rxn to 80% Dilution. Buffer to apply the load. The flow rate forthe sample application phase of the chromatography was 10 ml/min. Thecolumn effluent conductivity is 20 milli-Siemens under the loadingconditions. Reverse phase HPLC analysis; 500 ul injections of columnpass through, indicated that all material(s) were binding under theloading parameters.

Following application of the entire refold reaction, the column waswashed with Buffer A, for 20 column volumes until a steady UV absorbancebaseline at 280 nm wavelength was obtained. Upon obtaining the steadybaseline, the pump proportioning was set to 10% Buffer B and the columnwashed for 1 CV before eluting the bound protein with a 15 CV gradientfrom 10% buffer B (starting condition) to 100% B. A symmetric peakeluted fairly soon following initiation of the gradient. Fractionscontaining protein as indicated by the UV trace were analyzed by Reversephase HPLC for target content. Analysis of the cation exchange proteinpool demonstrated that a much higher proportion of dimeric product peakwas present at this stage, with some of the cluster peaks stillobservable. Non-reducing SDS-PAGE analysis on protein containingfractions was employed to look at their complexity. The gels indicate asignificant amount of monomeric and dimeric species, at a 1:1 ratio, arepresent at this stage.

Phenyl HP HIC Step:

The cation exchange pool is concentrated to 40 ml volume against a 10 kDcutoff membrane (Amicon Ultra-15) and adjusted to 0.7M [NH₄]₂SO₄ throughslow addition of sufficient solid to the well stirred concentrate.Finally, the pH was adjusted to 7.5 with 2N NaOH in preparation for anIsocratic passage over a hydrophobic interaction column. A highperformance Phenyl HP (Pharmacia) column (17 mL, 2 cm. dia.) wasequilibrated in 0.7M Ammonium Sulfate; 20 mM NaPO₄, 20 mM MES, pH 7.5buffer, at room temperature. The adjusted protein pool was applied tothe column using a superloop injection port at a flow rate of 2.5mL/min. Little protein passed during sample application at 2.5 ml/min.Once the entire sample had been injected, the flow rate of equilibrationbuffer was increased to washout unbound protein. However, uponincreasing the flow rate to 10 mL/min. for the wash phase, proteinstarted to elute from the column. A fairly symmetric peak eluted withsmall plateau's on the leading and trailing flanks. A pool, excludingthe leading and trailing plateau fractions was made and analyzed by RPHPLC. SDS-PAGE analysis (non-reducing) revealed that all of the higherMW multimers were cleared by this step, and that only monomer anddimeric species of IL17A remained, with the dimer being the predominantspecies.

Desalting Step:

The pooled protein from the hydrophobic chromatography step wasconcentrated to 10 mL against a 10 kD cutoff membrane (Amicon Ultra-15)and injected onto a Hi Trap 26/10 desalting column (Pharmacia)equilibrated in 35 mM NaPO₄ 109 mM NaCl, pH 7.0 buffer. Proteincontaining eluate fractions were analyzed by RP HPLC, sterile filtered,analyzed for protein content and tested for Endotoxin prior to beingaliquoted and frozen at −80 degrees

Example 51 Purification of Human IL17A CH6 from 293F B Cells

Recombinant carboxyl terminal 6-Histidine tagged human IL17A protein wasproduced from transfected CHO cells expressing the target atapproximately 0.3 mg/L. The CHO transfections were performed usingmethods known in the art. Approximately 7 L of conditioned media washarvested and sterile filtered using a 0.2 μm filter, concentrated to1.0 L liters in a Millipore ProFlux M12 tangential flow filtrationsystem equipped with two Pellicon 2 Mini 10K filters, then bufferexchanged with 10 volumes into PBS (0.137M NaCl, 0.0027 M KCl, 0.0072Na₂HPO₄, 0.0015 M KH₂PO₄, pH 7.4.) Protein was purified from this bufferexchanged media by a combination of HisTrap HP Immobilized MetalAffinity Chromatography (IMAC, GE Healthcare, Piscataway, N.J.,) andSuperdex 200 (GE Healthcare) Size Exclusion Chromatography (SEC.)

The 1.4 L buffer exchanged media was adjusted to 25 mM Imidazole andloaded overnight at 4° C., 1.2 ml/min (36 cm/hr) to a 5 ml (1.6×2.5 cm)HisTrap HP column pre-eluted with three CV 500 mM Imidazole andequilibrated with 20 CV 25 mM Imidazole, 50 mM Sodium Phosphate, 500 mMNaCl, 0.02% Azide pH 7.4. After loading, the column was washed with 20CV equilibration buffer, then eluted in two steps for 10 CV at 5 ml/min(149 cm/hr) with 50 mM Imidazole, and 500 mM Imidazole, both in 50 mMSodium Phosphate, 500 mM NaCl, 0.02% Azide pH 7.4. Eluted fractions wereanalyzed by SDS-PAGE, and those containing the target were pooled.Anti-His western analysis of pass-through indicated that all targetbound.

Next the 9 ml IMAC pool was concentrated to 0.9 ml and injected to a 23ml Superdex 200 (10 mm×300 mm) column equilibrated in 35 mM Phosphate,120 mM NaCl, pH 7.3 and eluted at 0.4 ml/min (30 cm/hr.) Fractions werecollected and pooled based on SDS-PAGE and A280, 0.2 μm filtersterilized and frozen as aliquots at −80° C. The concentration of thefinal purified protein was determined by BCA assay (Pierce, Rockford,Ill.). The overall process recovery was about 80%.

Analysis of Purified Human IL17A CH6

Recombinant Human IL17A CH6 was analyzed by SDS-PAGE (10% BisTris,Invitrogen, Carlsbad, Calif.) with 0.1% Coomassie R250 staining forprotein and after transfer to nitrocellulose, by immunoblotting withAnti-His-HRP. The purified proteins were electrophoresed using anInvitrogen Novex's Xcell II mini-cell, and transferred to nitrocellulose(0.2 mm; Invitrogen, Carlsbad, Calif.) at ambient temperature at 600 mAfor 45 minutes in a buffer containing 25 mM Tris base, 200 mM glycine,and 20% methanol. The filters were then blocked with 10% non-fat drymilk in 50 mM Tris, 150 mM NaCl, 5 mM EDTA, 0.05% Igepal (TBS) for 15minutes at room temperature. The nitrocellulose was quickly rinsed, andthe Anti-His-HRP antibody (1:2500) was added. The blots were incubatedovernight at 4° C., with gentle shaking. Following the incubation, theblots were washed three times for 15 minutes each in TBS, and thenquickly rinsed in H₂O. The blots were developed using commerciallyavailable chemiluminescent substrate reagents (Roche LumiLight), and thesignal was captured using Lumi-Imager's Lumi Analyst 3.0 software(Boehringer Mannheim GmbH, Germany.)

The purified Human IL17A CH6 appeared as 2-3 bands on both the Westernblot and the Coomassie stained gel under reducing and non-reducingconditions, suggesting glycosylated forms with the major band at about39 kDa under non-reducing conditions. The protein had the correct aminoacid composition and N-terminal sequencing yielded a strong singlesequence having the correct NH2 terminus.

Example 52 Human IL-17F Protein Production from E. coli: A1275F Process

Preparation and Extraction of Insoluble IL17F from Escherichia coli:

E. coli W3110 cells from a 2 L bioreactor fermentation containing thehuman IL17F were washed with 50 mM Tris pH 8 containing 200 mM NaCl and5 mM EDTA to remove any broth contaminants. 1.2 L of ice cold lysisbuffer (50 mM Tris pH 8, 200 mM NaCl, 5 mM EDTA, 5 mM Benzamidine and 5mM DTT) was added to the 350 g cell pellet and homogenized using thePolytron tissue-grinder until all clumps were disrupted. Next, thebacterial cells were lysed with three passes through a MicroFluidizerkeeping the cell suspension chilled to 4° C. The final volume of thecell lysate was 1.73 L. The inclusion bodies in this lysate werepelleted by centrifuging 30 min at 20,000×g (12,000 rpm in a JA-14 rotorin a Beckman J2-21M centrifuge), 4° C. in eight 250-ml centrifugebottles. The inclusion bodies were washed three times with lysis bufferto remove any E. coli unbroken cells and large cellular debris from thepelleted inclusion body protein. The supernatant was carefully pouredoff the pellet and the inclusion bodies were washed by suspending thepellet in lysis buffer and completely homogenizing to wash out solubleproteins and cellular components. The washed inclusion bodies wererecovered by centrifuging 30 min at 15,000×g (12,000 rpm in JA-14rotor), 4° C. One half of the washed inclusion bodies (˜64 grains wetweight) were stored at −80° C. in 250 ml bottles while the other halfwas processed further. 7M Guanidine-HCl in 50 mM Tris pH 8 containing100 mM sodium sulfite and 20 mM sodium tethrathionate was used toextract recombinant protein from washed pellets. Extraction with thedenaturant dissociates protein-protein interactions and unfolds theprotein. As a result, the extracted protein consists of unfoldedmonomers, with sulfhydryl groups in the reduced state and sulphonated.Using the tissue homogenizer, the 64 g pellet was homogenized with 100ml extraction buffer and clarified by centrifuging the suspension fortwo hours at 35,000×g at 4° C. The 60 ml of clarified extract wasevaluated by RP HPLC (28.4 mg/ml) and divided into four parts. Threeparts were stored at −80° C. until required. The fourth part was usedfor preparing refolded IL17F.

Refolding:

Ice cold refolding buffer (4 Liters) of the following composition wasprepared; 0.75M Arginine, 55 mM MES (N-MorpholinoEthaneSulfonic acid),10.56 mM NaCl, 0.44 mM KCl, 0.055% Peg 3.4 K (w/v), 1.1 mM EDTA, 440 mMSucrose, 550 mM GuHCl, 1 mM GSH (reduced Glutithione), 1 mM GSSG(oxidized Glutithione), pH 6.5

The oxidation-reduction pair (GSH:GSSG) was added just prior to dilutingthe GuHCL solubilized S-sulphonated inclusion body stock. Theconcentration of the Sulfytolized inclusion bodies was 28.4 mg/mL asdetermined by RP HPLC. 15 mL of the concentrated stock was added,dropwise, to 4 Liters of well stirred ice cold refolding buffer. Uponcompleting the dilution process, the vessel was tightly capped andplaced in the cold room with gentle stirring. At various time intervals,samples were withdrawn for RP HPLC analysis. The vessel was gentlystirred in the cold room for 72 hours before initiating recovery basedon HPLC analysis.

UF/DF Process:

Following refolding, the refold mixture was put through a UF/DF processphase. The mixture was first concentrated, Vs 5K cutoff membrane, from 4Liters down to approximately 475 mL. At this stage, the concentrate wasleft gently stirring in the cold room overnight. In the morning aDiaFiltration phase was initiated transitioning the retentate buffercomposition with 900 mL throughput of 20 mM Tris; 120 mM NaCl@pH 8.0buffer. As diafiltration proceeded, at fixed vessel volume of 475 mL,clouding and flux rate reduction was apparent. The contents of thevessel were harvested at this point and centrifuged to remove theprecipitate that had formed. RP HPLC analysis of the clarified solutionindicated that no target had precipitated. The clarified mixture wasdiluted 1:1 (v/v) with 25 mM Acetic acid@pH 5.4. The conductivitymeasured 9.8 milli-Siemens matching that which was needed for binding toa cation exchange column step. An additional 0.68 ml acetic acid wasadded, dropwise with simultaneous titration (addition 2N NaOH) to keeppH at 5.4. Thus adjusted solution was filtered through a 1.2 micronfilter and applied to the cation exchange column capture step.

Capture and Recovery on Cation Exchange (CIEX):

The adjusted solution from the UF/DF process was loaded at 20 mL/min toa 59 ml bed (2 cm dia.) of SP Fast Flow cation exchange media(Pharmacia) equilibrated in Buffer A: 25 mM Acetic acid; 100 mM NaCl@pH5.4. After completing the sample load, the column was washed with 20 CVBuffer A until a steady baseline absorbance at 280 nm was attained. Atthat point, the product was eluted with a 20 CV gradient formed betweenBuffer A and Buffer B: 20 mM acetic acid; 2M NaCl@pH 5.4. The gradientstarts at 100% Buffer A and at 20 CV is completed at 25% BufferA:75%BufferB. The CIEX process was run at room temperature. The producteluted as a symmetric peak between 30-60 milli-Siemens conductivity.Fractions containing protein were analyzed by non-reducing SDS-PAGEcoomassie staining and fractions with the dimeric IL17F were pooled andconcentrated in preparation for size exclusion polishing and bufferexchange.

Size Exclusion Chromatography:

The concentrated cation exchange pool (7 mL, containing 200 mg protein)was injected to a bed of Superdex 75 (26/60) size exclusion column(Pharmacia) equilibrated in 50 mM NaPO₄, 109 mM NaCl at pH 7.2 flowingat 3.5 ml/min. Eluted fractions containing mostly dimeric pure IL17Fprotein were pooled, sterile filtered, assayed for protein concentrationand endotoxin levels before being aliquotted and stored at −80 degrees.

From the foregoing, it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

1. An isolated polynucleotide encoding an antibody or antigen-bindingfragment thereof which specifically binds to a polypeptide of SEQ IDNO:4, wherein the antibody or antigen-binding fragment thereofcomprises: a) a light chain variable region comprising: i) a light chainCDR1 comprising amino acid residues 23 to 36 of SEQ ID NO:1014; ii) alight chain CDR2 comprising amino acid residues 52 to 58 of SEQ IDNO:1014; and iii) a light chain CDR3 comprising amino acid residues 91to 102 of SEQ ID NO:1014; and b) a heavy chain variable regioncomprising: i) a heavy chain CDR1 comprising amino acid residues 31 to35 of SEQ ID NO:1015; ii) a heavy chain CDR2 comprising amino acidresidues 50 to 66 of SEQ ID NO:1015; and iii) a heavy chain CDR3comprising amino acid residues 99 to 112 of SEQ ID NO:1015.
 2. Anexpression vector comprising the following operably linked elements: a)a transcription promoter; b) a DNA segment comprising the polynucleotideof claim 1; and c) a transcription terminator.
 3. A host cell comprisingthe expression vector of claim
 2. 4. A method of producing an antibodyor antigen-binding fragment thereof comprising: culturing a cellaccording to claim 3 under conditions wherein the antibody orantigen-binding fragment encoded by the DNA segment is expressed; andrecovering the antibody or antigen-binding fragment.